[VI]. Post-Transcriptional Processing and Post-Transcriptional Control of Gene Expression

• Processing of eukaryotic pre-mRNA About 60% of human genes give spliced mRNAs Eukaryotic cells have evolved RNA surveillance mechanisms

that prevent incorrectly processed RNAs to be transported out of the nucleus

• Regulation of pre-mRNA processing

• RNA editing

• Macromolecular transport across the nuclear envelop

• Cytoplasmic mechanisms of post-transcriptional regulation

• Processing of rRNA and tRNA

Overview of Post-Transcriptional Control of Genes

Processing of Eukaryotic Pre-mRNA

1. Capping of mRNA2. Splicing of mRNA3. Polyadenylation of mRNA

Overview of mRNA Processing in Eukaryotes

Processing of pre-mRNA is co-transcriptional

Structure of the 5’ Methylated CAP

• A methyl group from S-adenosyl-methionine is added to the N7 position of the G and the 2’ oxygen of the 5’ ribose at the nascent RNA

Synthesis of 5’-Cap on Eukaryotic mRNAs

• Capping occurs shortly after initiation of transcription

• 7-methyl-G is added in the 5’ end of the nascent RNA shortly after transcription initiates, about 25-30 nucleotides in length.

• The enzyme involved in this process is a dimeric capping enzyme associated with the phosphorylated carboxyl-terminal domain (CTD) of Pol II. Capping is specific for transcripts produced by Pol II

• The -phosphate is removed from the nascent RNA, replaced with a GMP (5’-5’ triphosphate structure), and a methyl group from S-adenosyl-methionine is added to the N7 position of the G and the 2’ oxygen of the 5’ ribose at the nascent RNA

• Capping of the nascent transcript is coupled to elongation so that all of the transcripts will be capped

• Capping of mRNA will protect it from degradation by 5’-exonuclease

Functions of Capping

• In prokaryotes, the Shine-Dalgarno sequence, localized at 10 bases upstream of AUG, of the polycistronic mRNA, that binds to 16S rRNA to initiate translation

• The AUG is localized within the consensus sequence of GCCA/GCCAUGG (Kozak’s sequence)

• In eukaryotes, the 5’ end of the mRNA is Capped. The CAP, after binding to the CAP-binding complex (CBC), will protect the mRNA from been degraded by RNase

• After been transported out of the nucleus, the CBC will be replaced with eIF4E and the complex will bind to 40S ribosome to initiate translation

• CBC contains RNA binding proteins, in mammals encoded by CBC20/CBC80 genes

• Following the initiation of transcription and of the first few bases of the RNA, the RNA polymerase II pauses and only continues transcription once the nacent RNA has been capped. Capping is essential for recruitment of the pTEF-b kinase which is required for transcriptional elongation

• GT: Guanylyl transferase

• MT: 7-methyltransferasse

Coupling Transcription with the 5’ Capping

RPB1 of pol II contains Tyr-ser-pro-Thr-Ser-Pro-Ser at the C-terminus

Polyadenylation

• Cleavage of RNA at the site downstream of AAUAAA and upstream of a sequence rich in G/U. These sequences are recognized by CPSF (cleavage- and polyadenylation-specific complex) and CstF (cleavage-stimulation factor)

• Endonucleolytic cleavage will take place

• Following that, polyadenylation will take place in the left fragment

• The right fragment will be degraded

3’ Cleavage and Polyadenylation of Pre-mRNA Are Tightly Coupled

• Eukaryotic mRNAs are polyadenlated except histone mRNA

• Poly(A) is added at 3’ end of the mRNA after endonuclease cleavage of the longer RNA transcript

• An AAUAAA sequence which is 10 – 35 nucleotide upstream of the poly(A) tail is the poly(A) signal

• Second Poly(A) signal (G/U rich or U rich sequence), about 50 nucleotides off the cleavage site, functions for efficient cleavage and polyadenylation

• CPSF (cleavage and polyadenylation specificity factor), a 360 kd complex consists of four different polypeptides

• CStF: cleavage stimulatory factor• CF: cleavage factor; PAP: poly(A) polymerase• PABPII: Stimulating polyadenylation

1

2

3

The 3′ mRNA End Processing Is Critical for Transcriptional Termination

1. RNA polymerase I and III terminate their transcription upon meeting a terminating signal on the DNA

2. The cleavage of pre-mRNA occurs at the site downstream of AAUAAA and upstream of a sequence rich in G/U. These sequences are recognized by CPSF (cleavage- and polyadenylation-specific complex) and cleavage-stimulation factor (CstF)

Polyadenylation Enhances the Stability of the mRNA

• Polyadenylation of mRNA will increase the stability of mRNA from degradation from the 3’ end

• In standard histone mRNAs without polyadenylation, the mRNAs are stablized by formation of hairpin loop during S phase

• Pre-mRNA during the processing are associated with many nuclear proteins, major components of hnRNPs (Heterogenous ribonucleoprotein particles)

• The nuclear RNA molecules are collectively referred as hnRNA (heternogenous nuclear RNA) – i.e., pre-mRNA and other nuclear RNAs of various sizes

• The hnRNA with the associated proteins can be visualized by immunostaining

• Proteins of hnRNP are with sizes of 34 to 120 kD. These proteins were isolated by irradiating the cultured cells to high-dose of UV, preparing nuclear extracts, run the extract through an oligo-dT cellulose column, recover the bound proteins, and then characterize the proteins.

• The hnRNP proteins have a modular structure: containing one or more RNA-binding domains and at least one domain that is believed to interact with other proteins

• A diverse set of proteins with conserved RNA-binding domains associate with pre-mRNAs

• Reading List VI: hnRNP Complex Heterogenous ribonuclear particles

hnRNP Proteins: a diverse set of proteins with conserved RNA binding domains associated with pre-mRNA

Functions of the hnRNP Proteins

• Interaction of the pre-mRNA with hnRNP proteins will prevent the formation of secondary structure by pre-mRNA, thus making the pre-mRNA accessible for interaction with other RNA molecules or proteins

• Pre-mRNA – hnRNP protein complex will make pre-mRNA a more uniform substrate for further processing

• hnRNP proteins A1, C, and D bind preferentially to the pyrimidine-rich sequences at the 3’ ends of introns

• The above observation suggests that different hnRNP proteins will bind to different RNA sequences that specify RNA splicing or cleavage/polyadenylation and contribute to the structure recognized by RNA-processing factors.

• Other studies suggest that hnRNP proteins may function in the transport of mRNA to the cytoplasm

Conserved RNA Binding Motifs (I) RRM Domain & Its interaction with RNA

• The RNA recognition motif (RRM), also called as RNP motif and the RNA binding domain (RBP) are the most common RNA-binding domains in the hnRNP proteins. This 80-residue domain contains two highly conserved sequences (RNP1 and RNP2) found in yeast to human

• RRM domain consists of 4-stranded β sheet flanked on one side by two α helices. The conserved RNP1 and RNP2 sequences lie side by side on the two central β strands and their side chains make multiple contacts with a single-stranded region of RNA.

RBD: RNA-binding domain containing 81 amino acid residues

Conserved RNA Binding Motifs (II)

• RGG Box: This is the other RNA-binding motif found in hnRNP proteins, containing five Arg-Gly-Gly (RGG) repeats with several interspersed aromatic amino acids

This motif is similar to the RNA-binding domains of the HIV Tat protein

• The KH motif: a 45-residue motif is found in the hnRNP K protein and several other RNA-binding proteins, commonly two or more copies of the KH motif are interspersed with RGG repeats

The 3D-structure of KH domain is similar to that of the RRM domain but smaller. It consists of three -sheet structure supported from one side by 2 -helices

RNA binds to the KH motif by interacting with a hydrophobic surface formed by the α helices and one β-strand

Splicing: RNA-DNA Hybridization to Introns Are Spliced from Pre-mRNA

• (a) Eco RI fragment of adenovirus DNA containing exon gene. The gene contains 4 short exons and three introns

• (b) Electron micrograph (left) and schematic drawing (right) of hybrid between DNA and RNA

• Introns in the pre-mRNA are removed by RNA splicing

• Richard Roberts and Philip Sharp were awarded with a Nobel prize in 1993 for the discovery of splicing of precursor mRNA

• For long transcription units, splicing of introns in the nascent RNA begins before the entire transcription is completed

• Reading List VI: (i). R Loop Mapping; (ii) Nobel Lecture by Richard Sharp; (iii) Mapping of viral RNA with viral DNA

Consensus Sequences around 5’ and 3’ Splice Sites in Vertebrate Pre-mRNAs

• Location of specific splice sites can be determined by comparing the genomic and specific cDNA sequences

• A pyrimidine rich region (~15 bases) is located in the upstream of the 3’ splice site

• About 30 – 40 nucleotides at each end of an intron are necessary for splicing to occur at normal rates

• Donor splice site: GU; Acceptor splice site: AG; This is termed GU/AG rule

Donor Acceptor

Assigned Reading: Intron splicing

Two Transesterification Reactions in Splicing of Exons

• Two sequential transesterification reactions are required to remove the intron sequence

• Introns are removed as a lariat-like structure in which the 5’ G of the intron is joined in an unusual 2’,5’-phosphoester bond to an adenosine near the 3’ end of the intron. This A is called the branch point.

• Since the number of the phosphodiester bond in the RNA molecule during splicing is not changed, the process does not require energy

Two Steps of Transesterification Reactions• First step of splicing reaction is

nucleophilic attack by the 2’-OH on the 5’ splice site

• The left exon takes the formof a linear molecule

• The right intron exon molecule forms a lariat, in which the 5’terminus generated at the end of the intron simultaneously transesterificates to become linked by a 2’-5’ bond to a base within the intron. The target base “A” in a sequence that is called the “branch site”

• In the second step, the free 3’-OH of the exon that was released by the first reaction attacks the bond at the 3’ splice site to form a 3’-5’ phosphoester bond

The consensus sequence of the branch site in yeast is UACUAAC, and in multicellular eukaryotes is purine and pyrimidines at each position

• U1 snRNP contains the core Sm proteins, three U1-specific proteins (U1 – 70K, U1A and U1C) and U1 snRNA

• U1 snRNA contains several domains: Sm-binding site: interacting

with the common snRNP proteins

Stem-loop structures: binding to proteins that are unique to U1 snRNP

• U1 snRNA interacts with the 5’ splice site by base pairing between its single-stranded 5’-terminus and a stretch of four to six bases of the 5’splice site

• Mutations in the 5’splice site and U1 snRNA can be used to test the

Structure of snRNA Molecule

importance of pairing of 5’ splice site and the snRNA

snRNA

snRNAs Base-Pairing with Pre-mRNA During Splicing

• Splicing requires the presence of small nuclear RNA (snRNA)• Five U-rich small nuclear RNAs (snRNA), designated as U1, U2, U4,

U5 and U6, participate in pre-mRNA splicing• These RNAs are 107 to 210 nucleotides long, associated with 6 to 10

proteins in small nuclear ribonucleoprotein particles (snRNPs)

• Small cytoplasmic RNAs (scRNA; scyrps) – RNAs that are present in the cytoplasm (and sometimes are also found in the nucleus).

• Small nuclear RNA (snRNA; snurps) – One of many small RNA species confined to the nucleus; several of them are involved in splicing or other RNA processing reactions.

• Small nucleolar RNA (snoRNA) – A small nuclear RNA that is localized in the nucleolus.

• The five snRNPs involved in splicing are U1, U2, U5, U4, and U6.

• Together with some additional proteins (splicing factors), the snRNPs form the spliceosome (~12 MDa).

• Figure in the left shows the number of proteins present in the sliceosome

Spliceosome

Formation of the Commitment Complex

• SR proteins: Proteins that play a critical role in the formation of spliciosomes. These proteins are splicing factors that contains one or two RNA recognition domains (RS domains; Arg/Ser repeats)

• U1 snRNP initiates splicing by binding to the 5′ splice site by means of an RNA–RNA pairing reaction.

• The commitment complex (or E complex) contains U1 snRNP bound at the 5′ splice site and the protein U2AF bound to a pyrimidine tract between the branch site and the 3′ splice site.

Intron Definition/Exon Definition (I)

• In yeast, all intron containing genes are interrupted by a single small intron, the 5’ and 3’ splice sites are recognized by U1 snRNP, BBP and Mud2. this is referred as “intron definition”

• The figure in the right shows exon definition which happens in short exons and long introns. More details are explained in the next slide

• In multicellular eukaryotic cells where splicing signals are highly variable, SR proteins play an essential role in initiating the formation of the splicing commitment complex

Intron Definition/Exon Definition (II)

• Intron definition mechanism also applies to splicing of small introns in multicellular eukaryotic cells

• Many muticellular eukaryotic genes possess long introns with many sequences resemble the real splice sites. To ensure correct recognition of the splice sites, the mechanism of “exon definition” is employed

• In exon definition, the U2AF heterodimer (U2 snRNP auxiliary factor) binds to the 3’splice site and U1 snRNP base pairs with the 5’ splice site downstream from the exon sequence. This process may be aided by SR proteins that bind to specific exon sequences between the 3’ and downstream 5’ specific sites. By an unknown mechanism, the splicing is done properly

• The commitment U4 dissociates from U6 snRNP to allow U6 snRNA to pair with U2 snRNA to form the catalytic center for splicing.

• Both transesterification reactions take place in the activated spliceosome (the C complex).

• The splicing reaction is reversible at all steps.

• E complex progresses to pre-spliceosome (the A complex) in the presence of ATP.

• Recruitment of U5 and U4/U6 snRNPs converts the pre-spliceosome to the mature spliceosome (the B1 complex).

• The B1 complex is next converted to the B2 complex in which U1 snRNP is released to allow U6 snRNA to interact with the 5′ splice site.

The Spliceosome Assembly Pathway

• The final step of transesterification is the formation of the lariate

Splicing Utilizes a Series of Base Pairing Reactions between snRNAs and Splice Sites

• U1 pairs with 5’ splice site

• U2 pairs with the branch site

• U6 pairs with 5’ splice site

• U5 is close to both exons

Mutation that Affects the Binding of SR Proteins to the Exonic Splicing Enhancer

• Mutation in SR protein that results in unable to bind to the exonic splicing enhancer-------resulting skipping exons during splicing. The truncated mRNA will be degraded or translated into protein with abnormal function

• For example, spinal muscle atrophy, a disease that causes childhood mortality, is resulted from mutation of SR protein that fails to bind to the exonic splicing enhancer of SMN2 pre-mRNA and causes exon skipping leading to low production of SMN2 protein.

• In childhood, low levels of SMN2 protein will lead to low viability of spinal cord motor neurons, resulting in death.

Approximately 15% of the single base-pair mutations that cause human genetic diseases interfere with proper exon definition. Although some of the mutation may lead to use of alternative splice sites, others will result in skipping exons due to SR protein failing to bind to the exonic splicing enhancer (a six- base motif)

Splicing Can Also Occur Between AU and AC

A minority of introns begin with AU and AC as opposed to the GU and AG found in most introns. Removal of these types of introns involves lariat formation and the U5 RNA, but the other U RNAs involved are different in the two cases. The AU-AC type splicing takes placing in the cytoplasm, suggesting that this type of splicing may have different function than the GU-AG type of splicing in the nucleus.

Hammerhead Ribozyme

• Thomas Cech discovered an RNA molecule that possessed enzymatic activity that could remove intervening sequence in tetrahymena rRNA– this discovery led to a Nobel prize in 1989

• Self Splicing Introns (Ribozymes) : Introns that can be spliced out in the absence of splicing protein factors in vitro

Discovery of a Hammerhead Ribozyme

• Reading List VI:(i) Cech’s Nobel Prize lecture(ii) Hammerhead ribozyme

Self-Splicing of Group II and Group I Introns

• Group I introns (present in nuclear rRNA genes of protozoans) and group II introns (present in protein-coding genes and some rRNA and tRNA genes in mitochoria and chloroplasts of plants and fungi)

• Group II introns excise themselves from RNA by an autocatalytic splicing event (autosplicing or self-splicing).

• The splice junctions and mechanism of splicing of group II introns are similar to splicing of nuclear introns.

• A group II intron folds into a secondary structure that generates a catalytic site resembling the structure of U6-U2-nuclear intron.

• Group I introns excise themselves from RNA also by a autocatalytic splincing event

Comparison of Self-Splicing Group II Introns and U snRNAs in Spliceosome

• Two types of self splicing introns have been discovered: Group I introns (present in nuclear rRNA genes of protozoans) and group II introns (present in protein-coding genes and some rRNA and tRNA genes in mitochoria and chloroplasts of plants and fungi)

First Trans-esterification

Second Transesterifi-cation

Self-Splicing Group II Introns Provide Clues to the Evolution of snRNAs

• Even though the precise sequences of group II introns are not highly conserved, they fold into conserved, complex secondary structure containing numerous stem loops

• The chemistry of self splicing by a group II intron is similar to that found in pre-mRNA. This observation led to a hypothesis that “snRNA function analogously to the stem-loop in the secondary structure of group II introns”

• According to this hypothesis, one expect to see the 3D structures presented in the previous slide

• Introns in ancient pre-mRNAs evolved from group II self-splicing introns through progressive loss of internal RNA structures, which concurrently evolved into trans-acting snRNA that perform the same functions

• Support for this type of evolutionary model comes from experiments with group II intron mutants in which domain V and I are deleted. The resulting mutant fail to perform self-splicing

• Maturase enzyme may function to stabilize the 3-D structure for self-splicing of group II introns in vivo

Nuclear Exonucleases Degrade RNA That Is Processed Out of Pre-mRNA

• The spliced introns are degraded by exonucleases from 5’ or 3’ end.

• The 2’,5’-phosphodieaster bond in the newly spliced intron is excised to linear structure by a debranching enzyme. The predominant decay pathway of the linear RNA molecule is 3’ to 5’ by 11 exonucleases that associate with one another in a large protein complex called the “exosome”

• Other protein in the exosome is RNA helicase that disrupt baser pairing and protein RNA interactions

• Exosome also functions to degrade improper processed or polyadenylated pre-mRNA

• Nuclear cap-binding complex: protein that bind to 5’cap to prevent the cap been destroyed by exonulceases

Chain Elongation by RNA Pol II Is Coupled to the Process of RNA-Processing Factors

• The carboxyl-terminal domain (CTD) of RNA Pol II is composed of multiple repeats of a seven residue sequence. When fully extended, the CTD domain in yeast enzyme for instance is about 65 nm long

• The CTD of human RNA Pol II is about twice as long• The long CTD allows multiple proteins to associate with a single RNA Pol II

molecule• For instance, enzyme that adds the 5’ cap to the nascent transcription

initiation as well as RNA splicing and polyadenylation factors are associated with phosphorylated CTD. As a consequence, these processing factors are present at high local concentrations when splice sites and poly(A) signals are transcribed by the polymerase, enhancing the rate and specificity of RNA processing. Deletion of CTD will reduce transcription and processing of RNA

• The association of RNA splicing factors with the phosphorylated CTD stimulates transcription elongation

Coupling of Transcription and RNA Processing

• Transcription and RNA processing are coupled in the nucleus: Capping Release pausing Splicing Polyadenylation

• Recruiting of these various factors is closely coupled to the phosphorylation of the CTD of RNA polymerase II (see next slide for details)

• CE: capping enzyme; SC: splicing complex; PC: polyadenylation complex; ph: phosphorylation

• Trimethylation of the arginine at position 4 in histone H3 not only results in a more open chromatin structure but also stimulates transcriptional elongation and enhances RNA splicing

• Both the CBC and the CPSF polyadenylation complex can interact with splicesome (S), so linking together these different post-transcription events

Methylation of Arginine at H3 Stimulates RNA Splicing

• Splicing can occur during or after transcription.

• The transcription and splicing machineries are physically and functionally integrated.

• Splicing is connected to mRNA export and stability control.

• Exon junction complex (EJC) – A protein complex that assembles at exon–exon junctions during splicing and assists in RNA transport, localization, and degradation.

Splicing Facilitate Transport of mRNA to cytoplasm

EJC: exon junction complex, recruiting several protein complex for mRNA transport

REF/Aly, a key protein mediating mRNA transport by interacting with TAP (Mex67p)

• Splicing in the nucleus can influence mRNA translation in the cytoplasm.

• Some aberrant mRNA in the cytoplasm that still has EJC associate with it, the EJC can recruit Upf that promotes decapping enzyme to remove CAP from the mRNA and leading degradation of the mRNA

• Nonsense-mediated mRNA decay (NMD) – A pathway that degrades an mRNA that has a nonsense mutation prior to the last exon.

The EJC Complex Couples Splicing with

NMD

• Regulation of Pre-mRNA Processing

• Macromolecular Transport Across the Nuclear Envelop

Regulation of Pre-mRNA Processing

• Alternative splicing is the principle mechanism for regulating mRNA processing By comparison of the genomic sequences of genes and the

sequences of the expressed sequence tags (ESTs) of cDNAs reveals that several genes have complex transcription units, and capable of producing several different mRNAs by different combinations of exons

More than 60 % of all transcription units in human genome are complex

Although cleavage at alternative poly(A) sites of pre-mRNA are known example, alternative splicing of different exons is the more common mechanism for expressing different protein from one complex transcription unit

Such alternative processing pathways are usually regulated in a cell-type specific or developmental stage-specific manner. Example: different fibronectin isoforms are produced in fibroblasts and hepatocytes

Different Mode of Alternative Splicing

• Specific exons or exonic sequences may be excluded or included in the mRNA products by using alternative splicing sites.

• Alternative splicing contributes to structural and functional diversity of gene products.

Effect of Alternative Splicing on Gene Expression (I)

• Alternative splicing can affect gene expression in the cell at least in two ways: Create structural diversity of gene products by including or omitting

some coding sequences or creating alternative reading frames for a protein of the gene. Example: CaMKII gene encoding kinases

Differentiatial splicing of the pre-mRNA for kinase results in production of three different kinases localized in different locations in the cells and kinasing different substrates

Another example is that alternatively spliced products may exhibit opposite functions. This example applies to all genes involved in apoptosis; one form promote apoptosis and the other form protect cells from apoptosis

Alternative Splicing of Primary Transcripts Where Both 5’ and 3’ Ends

of Transcripts Are Identical

In muscle troponin T gene, the same precursor mRNA can be spliced in up to 64 different ways in different muscle cell types due to the presence of tissue-specific splicing factors

The indication of the presence of tissue specific splicing factors for differential removal of exons 4-8 in different muscle cells comes from the following experiment. When muscle troponin T gene is expressed in non-muscle cell types, exons 4-8 are completely removed and resulting in only one mature mRNA.

Expression of Dscam Isoforms in Drosophila Retinal Neurons

• The most extreme example of regulated alternative RNA processing is the expression of Dscam gene in Drsophila

• Dscam gene encodes a set of proteins in the neuron of Drosophila

• Mutations in this gene interfere with the normal connections made by the axons of the retinal neurons with neurons in a specific region of the brain

• There are 95 alternatively spliced exons that could be spliced to generate 38,000 possible isoforms

• These results raise the possibility that the expression of different Dascam isoforms through regulated RNA splicing helps to specify the tens of thousands of different specific synaptic connections made between retinal and brain neurons

• In other words, correct wiring of neurons in the brain may depend on regulated RNA splicing

Expression of Ca++-Activated K+ Channel Isoforms mRNAs in Vertebrate Hair Cells Is Another Example

• In the inner ear of vertebrates, individual “hair cells” which are ciliated neurons, respond most strongly to a specific frequency of sound

• In birds and reptiles, the turning of hair cells is affected by the opening of K+ channel in response to Ca++ concentration changes. The channel opens determine the frequency with which the membrane potential oscilates, and hence the frequency to which the cell is turned

• Slo gene controls the channel, which is expressed in multiple, alternatively spliced mRNAs.

• Suggested Reading List Vi:Distribution of Ca++-activated K+

chennel isoforms along the tonotopic gradient

• Slo proteins encoded by different alternative spliced mRNAs open Ca++-activated K+ channel at different Ca++ concentrations

• Hair cells with different response frequencies express different Slo channel protein depending on their position along the length of the cochlea

• There are 8 regions on the slo mRNA where alternative exons are utilized, permitting the expression of 576 possible isoforms

• RT-PCR analysis of sol mRNAs from individual hair cells has shown that each hair cell expresses a mixture of different alternative spliced sol mRNAs with different forms predominating in different cells according to their position along the cochlea

• In rat, the splicing at one of the alternative splice sites in the slo pre-mRNA is suppressed when a specific protein kinase is activated by depolarization of the neurons. This observation suggests that a splicing repressor specific for this site may be activated when it is phosphorylated by this protein kinase

• These observations suggest that modification of splicing factors may play a significant role in modulating neuron function

• Suggested reading List IV:– Distribution of Ca++-Activated K+ Channel Isoforms along the Tonotopic

Gradient

Effect of Alternative Splicing on Gene Expression (II)

This figure shows models by which an alternative splicing factor can affect splicing by binding to a cis-acting element. In (a), the factor acts by promoting the use of the weaker of the two potential splicing sites while in (b) it acts by inhibiting use of the stronger of the two sites so that other, weaker, site is used.

The sex determination in Drosophila is the best characterized example

Alternative splicing may affect various properties of the mRNA by including or omitting certain regulatory RNA elements, which may significantly alter the half-life of the mRNA. This form of the splicing is caused by the presence of a splicing factor

Example: Regulation of Alternative RNA Splicing Leading to Sexual Differentiation in Drosophila

• The earliest example of regulated alternative splicing of pre-mRNA came from studies of sexual differentiation in Drosophila

• Sxl protein, encoded by sex-lethal gene, is produced in female Drosophila by a promoter only functions in female embryos. Later in embryonic development, this female-specific promoter is turned off, and a promoter is tuned on in both male and female embryos. In the absence of Sxl protein in male embryos, the sex-lethal pre-mRNA is processed into a mature mRNA with a stop codon in the sequence and thus produce no functional Sxl protein

• Sxl protein directs splicing of sex-lethal pre-mRNA to produce functional Sxl mRNA

Cascade of Regulated Splicing that Controls Sex Determination via Expression of Sex-Lethal (sxl) Transformer

(tra) and Double-Sex (dsx) Genes in Drosophila

• The earliest example of regulated differentiation splicing of pre-mRNA come from studies of Drosophila sexual differentiation

• Two proteins were found to regulate differential splicing of pre-mRNA, one of which is Sxl protein and the other is Tra protein

• Sxl protein regulates alternative RNA splicing of the Sxl mRNA in early embryonic development of females but not in males

• Sxl protein also regulate splicing of transformer gene pre-mRNA in females by the similar mechanism and the Tra protein is produced

• In female embryos, Tra protein and two other constitutive expressed proteins, Rbp1 and Tra2, directs splicing of exon 3 to exon 4 of the double-sex pre-mRNA and also promotes cleavage/polyadenylation at the alternative poly (A) site at the 3’ end of the exon 4 and thus producing female specific Dsx protein

• In male embryos, due to the absence of Tra protein, a different Dsx protein is produced which functions as a repressor to inhibit the expression of genes essential for female development in male embryos. Conversely female Dsx protein inhibits the expression of genes for male development in female embryos

Splicing Can Be Regulated by Exonic and Intronic Splicing Enhancers and Silencers

• Alternative splicing is often associated with weak splice sites

• Sequences surrounding alternative exons are often more evolutionarily conserved than sequences flanking constitutive exons

• Specific exonic and intronic sequences can enhance or suppress splice site selection

• SR proteins are the known proteins binding to ESE; hnRNP A/B proteins are known to bind to ESS; RBP proteins are known to bind to ISS or ISE

• The effect of splicing enhancers and silencers is mediated by sequence-specific RNA binding proteins, many of which may be developmentally regulated and/or expressed in a tissue-specific manner.

• The rate of transcription can directly affect the outcome of alternative splicing.

• Nova and Fox families of RNA binding proteins can promote or suppress splice site selection in a context dependent fashion. Binding of Nova to exon or flanking upstream introns inhibits the inclusion of alternative exon while Nova binding to the downstream flanking intron sequences promotes the inclusion of the alternative exon.

• Fox binding to the upstream intron inhibits the inclusion of the alternative exon and vice versa

The Nova and Fox Families of RNA Binding Proteins

Splicing Repressors and Activators Control Splicing at Alternative Sites

• Sxl protein prevents splicing, causing exons to be skipped whereas Tra promotes splicing. Therefore, Sxl protein is considered as a repressor for splicing and Tra protein is considered as an activator for splicing

• In the processing of the fibronectin pre-mRNA, Sxl like repressor could be expressed in the human hepatocytes to bind to the splice sties of the EIIIA and EIIIB exons in the fibronectin pre-mRNA, causing them to be skipped during splicing. On the other hand, a Tra-like protein could be expressed in the fibroblasts and resulting in inclusion of EIIIA and EIIIB in the fibronectin mRNA

RNA Editing Alters the Sequences of the Pre-mRNAs

• RNA editing: the sequence of the pre-mRNA is altered; and as the result, the sequence of the corresponding mature mRNA differs from the exons encoding it in the genomic DNA

• RNA editing is widespread in the mitochondria of protozoans and plants, and also in chloroplasts

• In higher vertebrates, RNA editing is much rarer, and mostly in single base changes

• ApoB gene in mammals is one of the example of RNA editing. In the liver, ApoB gene is transcribed into a longer mRNA which encodes a protein with two domains (green domain binds to lipid and the orange domain binds LDL receptor). In the intestine, the CAA codon of ApoB is edited to UAA [a stop codon] so the protein [ApoB-48] can bind lipid but not with LDL receptor

RNA Editing Involves a Change from an A to an I

Besides C- to U- editing, A- to I- editing has also been observed in human

An editing of A- to I- residue in the transcript of a receptor for the excitatory amino acid glutamate in neuronal cells results in encoding an arginine rather than glutamine, leading to the receptor permeable to calcium

Animals with this editing yet lacking ADAR2 (adenosine deaminase enzyme) will result in death

Effect of A- to I- RNA Editing and RNA Splicing

A- to I- editing of ADAR2 mRNA can also affect the splicing of this mRNA as well as the encoded protein

Another effect of A- to I- editing can also target micro RNA (miRNA) by affecting it binding to the target RNA (The function of miRNA will be discussed later)

Effect of A- to I- Editing Affects the Function of miRN A

A- to I- editing in miRNA376 will result in a tissue specific effect in mice

It results in inhibiting a different set of genes in kidney but not in the liver

In conclusion, the existence of two distinct types of editing enzymes, adenosine and cytidine deaminases, in mammalian cells each with multiple substrates indicates that this process represents a widely used mechanism which like alternative splicing that can produce related but distinct proteins from the same gene

Macromolecular Transport Across the Nuclear Envelop

• Messenger Ribonuclear Protein Complex (mRNP): mature mRNA complexed with specific hnRNP, the complex will be transported out of the nucleus

• The nucleus is separated by a double membrane (nulcear envelope) from the cytoplasm

• Mature mRNPs, tRNAs, ribosomal subunits are transported out of the nucleus through nuclear pores, and transport of nuclear proteins from cytoplasm into nucleus is also through the same route

• Small molecules and globular proteins >60 kDa can diffuse directly through the pores

• Large proteins and ribonucleoproteins are transported by the aid of transporters

Large and Small Molecules Enter or Leave the Nucleus via Nuclear Pore Complex

• Nuclear Pore Complexs (NPCs): Large symmetrical structures composed multiple copies of approximately 30 different nuclear proteins

• Under EM, the nuclear pore complexes showed a octagonal, membrane-embeded structure from which eight (~100 nm-long) filaments extend into the nucleoplasm. The distal ends of these filaments are joined by the terminal ring forming a nuclear basket

• The membrane embeded protein is also attached directly to nuclear lamina

• The cytoplasmic filaments extend from the cytoplasmic side of the NPC to the cytosol

• Four proteins are required for transporting macromolecules into the nucleus have been purified. These proteins are: Ran, nuclear transport factor 2 (NTF2), importin , importing

• Ran is a monomeric G protein, existing in two conformations, one can complexe with GTP and another one with GDP

• Importin and importing form a heterodimeric nuclear-import receptor, the submit bind to the NLS (nuclear localization signal) and the -submit binds to a class of nuclearproteins (FG-nucleoproteins). These nucleoproteins line the channel of the nuclear pore complex and are also found in the nuclear basket and the cytoplasmic filaments.

• FG-nucleoproteins contain multiple short repeats rich in phenylalanin (F) and glycine (G) residues

• Recently several importin analogs have been identified which can transport proteins with other class of NLS into nucleus without the need to bind with importin

FG-Domains of FG-Nucleoproteins

The FG-domains of FG-nucleoproteins

The FG-repeats are thought to associate with each other reversibly and rapidly forming a constantly re-arranging molecular sieve that allows diffusion of small water-soluble molecules through it

mRNA Exporter Helps Export of mRNP’s from the Nucleus to Cytoplasm

• Temperature sensitive mutation studies in yeast revealed that a heterodimeric mRNA-exporter is required for export of mRNPs out of nucleus

• mRNP exporter (TAP) is a heterodimer consisting of a large submit (NXF1) and a small submit (NXT1). TAP binds to mRNP through association with RNA and other proteins in the mRNP (REF, RNA export factor)

• The TAP/NXT1 mRNP also associate with SR proteins (splicing factors) that will direct both splicing and transport

• Small submit of mRNA-exporter is

homologous to NTF2 (nuclear transport factor 2), and interacts with a region of the larger submit that also shares homology to NTF2. Together, they form a domain that interacts with FG repeats in FG-nucleoproteins similar to NTF2 which is a dimer

• A C-terminal domain of the mRNA exporter also interacts with FG-repeats

Remodeling of mRNPs During Nucleat Export

• Some mRNP proteins dissociate from the nuclear RNP complex before export through an NPC while the others are exported to the cytoplasm and dissociated from the mRNP and shuffled back to nucleus

• These proteins include NXF1/NXT1 mPNP exporter, cap-binding complex (CBC), and PABPII

Reversible Phosphorylation and Direction of mRNP Nuclear Export

• Results of recent studies indicated that direction of mRNP export from the nucleus into the cytoplasm is controlled by phosphorylation and dephosphorylation of mRNP adaptor proteins such as REF

• In yeast, SR protein (Npl3) functions as an adaptor protein that promotes binding of the yeast mRNP exporter. Phosphorylation and dephosphorylation are involved

• 1. Phosphorlated NpI3 binds to nascent pre-mRNA

• 2. Following polyadenylation, the NpI3 is dephosphorylated and

promoting the binding of NXF1/NXT1 the mRNP complex•3. The mRNP exporter allows transport of mRNP•4. Sky (cytoplasmic kinase) phosphorylates NpI3 in the cytoplasm•5. Dissociaation of NXF1/NXT1 and NpI3 from the mRNP and return to nucleus

Other Proteins Assist mRNP Transport

• Some mRNP proteins assist binding of mRNA with mRNA-exporter SR proteins Exon junction complex Nuclear cap binding complex Poly (A)-binding protein (PABPI) Other RNA-binding protein

Nuclear Export of Balbiani Ring mRNPs

• The salivary glands of larvae of the Chironomous tentans has large RNA containing chromosome puffs called Balbiani Ring containing RNA-protein complex. These giant RNPs contain mRNA (75 kb) of glue protein

• After processing, the pre-mRNA in Balbiani ring in the form of RNP migrate through the nuclear pores

Formation of Hetergenous hnRNPs and Export of mRNPs

Single chromatin transcription loop containg assembled Balbiani ring RNP

The schematic drawing of the biogenesis of hnRNP

Model for the Transport of BR mRNPs Through the Nuclear Pore Complex (NCP)

But pre-mRNAs in spliceosomes are not exported from the nucleus

Transport of the Balbiani Ring RNP across the Nuclear Pore Complex (NPC)

Fusion of a Nuclear Localization Signal (NLS) to a Cytoplasmic Protein Causes the Protein to Enter

the Cell Nucleus• Nuclear-localization

signal (NLS) is the sequence found in large T-antigen of simian virus (SV40)

• The particular sequence is Pro-Lys-Lys-Arg-Lys-Val at the C-terminal end of the large T-antigen

• Fusion of the NLS sequence to other cytoplasmic protein will result in transport of the protein to the nucleus

• Unlike the NLS of T-antigen, an NLS in the hnRNP A1 protein is hydrophobic

Mechanism for Nuclear Import of “Cargo” Proteins

The details of the transport are described in next slide

• Free monomeric importin forms complex with the cargo protein by binding to the NLS of the cargo in the cytoplasm

• The cargo-importin complex enters the nucleus through the nuclear pore complex as the importin binds to FG-proteins

• In the nucleoplasm, the importin binds to Ram-GTP to form Ran-GTP-importin complex and the cargo is released in the nucleoplasm

• The Ran-GTP-importin complex diffuse through the NPC to the cytoplasm

• In the cytoplasm, importin interacts with a specific GTPase-accelerateing protein (Rn-GAP) that is a component of the NPC cytoplasmic filaments, This stimulates Ran to hydrolyze its bound GTP to GDP causing Ran has low affinity to importin

• The importin will participate in another cycle of transporting new cargo, while the Ran-GDP will return to nucleus by nuclear transport factor 2 (NTF2) and a guanine nucleotide-excchange factor (GEF) will cause release of GDP and rebinding to GTP

• The entire transport is driven by a facilitated diffusion model

Ran-Dependent Nuclear Transport

• NES: Nuclear export signal on the cargo

• Exportin (nuclear export receptor): Nuclear protein binding to cargo directed for export from nucleus to cytoplasm

• Export of molecule out of the nuclear requires Ran-GTP

Ran-Independent Nuclear Transport

• Most mRNAs are transported from the nucleus by a Ran-independent mechanism

• mRNA exporter: a heterdimer of TAP/NXT1

• This form of transport does not require Ran-GTP

• Cytoplasmic mechanisms of post-transcriptional regulation

Regulatory RNAs

Localization of mRNAs and their stability

Global regulation fo protein synthesis

• Processing of rRNA and tRNA

Regulator RNA

• RNA functions as a regulator by forming a region of secondary structure (either inter- or intramolecular) that changes the properties of a target sequence.

• The formation of double stranded structure between regulator RNA and the target mRNA will have two consequences:

Formation of the double-helical structure may itself be sufficient for regulatory purposes (preventing translation or degradation by ribonucleases)

Duplex formation may be important because it sequesters a region of the target RNA that would otherwise participate in some alternative secondary structure

A Riboswitch Can Alter Its Structure According to Its Environment

• A riboswitch is an RNA whose activity is controlled by the metabolite product or another small ligand (a ligand is any molecule that binds to another).

• A riboswitch may be a ribozyme.

• Aptamer – An RNA domain that binds a small molecule; this can result in a conformation change in the RNA.

• An enzyme encoded by glms which catalyzes the synthesis of GlcN6P (glucosamine 6 phosphate) from fructose-6-phosphate

• GlcN6P binds to the ribozyme sequence in the 5’ UTR leading to cleavage of the mRNA

• Vast tracts of the eukaryotic genome are transcribed.

• Antisense gene – A gene that codes for an antisense RNA that has a complementary sequence to an RNA that is its target.

• Antisense RNA – RNA that has a complementary sequence to an RNA that is its target.

Noncoding RNAs Can Be Used to Regulate Gene Expression

• Therefore, antisense gene can be used to turn off the expression of a gene at will

• Nested genes: Genes located within the introns of other genes. About 10-15% of genes have nested genes. If these genes are transcribed from the opposite strand, antisense RNAs are produced. The head to head arrangement of a nested gene will lead to transcriptional interference (TI).

• Transcriptional interference is emerging as a significant mechanism of transcriptional regulation, is the origin of the interference RNA

Antisense RNA Regulates Gene Expression.• Recent studies showed that

about 70% the human genes produce anti-sense RNA. This patent various with the cell types. In addition, DNA in the intergenic regions are also transcribed. These RNA products can result in non-coding RNAs with regulatory function

• In yeast, gene PHO84 is regulated in part by a class of non-coding RNAs named “cryptic unstable transcripts” (CUTs) transcribed from a promoter on the non-coding strand of PHO84

• Under the normal condition, this transcript is unstable and rapidly degraded by TRAMP and exosome RNase complex

• In the absence of degradation or in aging cells, the anti-sense RNA persists. It recruits histone deascetylase to remove acetyl group from the acetylated histone and thus causes remodeling of chromatin and turns off the gene activity

• In human, PROMPTs (promoter upstream transcripts) may have the same function

TRAMP complex (Trf4/Air2/Mtr4p Polyadenylation complex) is a protein complex consisting of the RNA helicase (Mtr4), a poly(A) polymerase (PAP) (either Trf4 or Trf5) and a zinc knuckle protein (either Air1 or Air2). The TRAMP complex interacts with the Exosome complex in the nucleus of eukaryotic cells and is involved in the 3’ end processing of ribosomal and snoRNAs (small nuclolar RNA)

TRAMP Complex

Bacteria Contain Regulator RNAs

• Bacteria contain up to hundreds of genes encoding RNA molecules ranging from 50 to 200 bp with regulatory functions. These RNA molecules are called regulator RNAs (sRNAs)

• Some of the sRNAs are common for several genes and others are specific to individual genes

• All sRNAs are imperfect anti-sense RNA molecules

• sRNAs function to (i) prevent transcription of the gene; (ii) affect processing of its RNA product; (iii) affect translation of the mRNA; and (iv) affect the stability of the mRNA by the formation of RNA-RNA duplex

• Example: Oxidative stress in E. coli is an example When E coli is exposed to reactive oxygen species, the expression

of antioxidant defense genes are induced. Hydrogen peroxide activates the transcription of OxyR which activates the transcription of several inducible genes including OxyS which encodes sRNA

OxyS gene product is a small RNA of 109 bases long. It is a trans-regulator that repress the translation of fihA mRNA

• OxyS RNA can also regulate rpoS, a gene coding for an alternative sigma factor. RpoS gene can also be positively regulated by two sRNA (dsrA and rprA). These three sRNAs are the global regulators that coordinate responses to environmental conditions

• Several of the sRNAs are bound by the protein Hfq, which increases their effectiveness

• The oxyS sRNA activates or represses expression of >10 loci at the post-transcriptional level

• We just to realize that many small RNAs in bacteria possess activities in controlling processes in the life cycle. Tandem repeats like CRISPR can be transcribed into powerful antiviral RNAs. The example is shown in next slide

Duplex formation between fihA mRNA and oxyS RNA

Effect of OxyS RNA on Translation of FihA mRNA

The CRISPRs System in E. coli

The CRISPRs (clusters of regulatory interspersed short palindromic repeats) locus in E. coli is transcribed into a large precursor RNA, which is processed by the cascade protein complex into short fragments that contain unique spacers identical to sequences in the phage DNA. Assisted by a protein,

Cas3, these small CRISP RNAs block the phage infection cycle. These mechanisms offer powerful approaches for turning off genes at will.

Cytoplasmic Mechanisms of Post-transcriptional Control (I)

• Micro RNA: was discovered in the nematode (C. elegans) during analysis of lin-4

and let-7 genes Cloning and nucleotide sequence analysis revealed that lin-4 and let-7

do not encode any protein product, but encoding RNAs of 21 and 22 nucleotides long, respectively. lin-4 RNA hybridizes to the 3’-untranslated region of lin-14 mRNA and lin-28 RNA and degrades the mRNA

About 100 different miRNA have been found in C. elegans and at least as many or more found in human. These miRNAs are used to degrade many mRNAs. MicroRNAs are transcribed by Pol II

All miRNA precursors are about 70 nucleotides long, and are processed by a ribonuclease (Dicer) to produce mature miRNAs of 21-22 bases

Base pairing between the miRNA and the 3’-untranslated region of an mRNA does not have to be 100% complementary. This differentiates it from the RNA interference. In human, more than 1,000 different miRNA are produced

Reading List VI: (i) Micro RNA (ii) Micro RNA controlling cell division – differentiation and cell death

RNAi Inhibits Transcription

• In an attempt to inhibit the translation of an mRNA in C. elgans by microinjecting single stranded antisense RNA, an unexpected result was found in the control experiment. In this control experiment, perfectly base paired double stranded RNA of several hundred bases long was microinjected into C. elegans, more effective inhibition was observed. Similar results were observed in plant system. This led to the discovery of siRNA (RNAi)

• Further studies revealed there are large number of double stranded transcribed from non-protein coding genes in the nucleus. These RNA molecules are processed into 21-23 bases long siRNA by enzyme systems similar to the processing of miRNA

• Nuclear proteins homologous to the cytoplasmic “Dicer” and “Argonatue protein generate nuclear siRNA complexes composed of different proteins from cytoplasmic RISC complexes. These nuclear siRNA complexes are thought to targeted to specific gene by base paring with nascent pre-mRNAs. This interaction induces the methylation of histone H3 at lysine 9, generating a binding site for HP1 proteins and the subsequent assembly of heterochromatin

Cytoplasmic Mechanisms of Post-transcriptional Control (II)

• RNA interference: RNA interference induces degradation of mRNAs with sequence

complementary to double-stranded RNAs RNA interference was discovered from initial attempts to inhibit the

translation of an mRNA by microinjecting a RNA with complementary sequence (antisense inhibition). In the control experiment, scientists found that perfectly base-paired double-stranded RNA of a few hundred pairs long was much more effective in inhibiting the expression of the gene than the anti-sense RNA alone

It was found that as long as one of the strand the ds-RNA is complementary to the mRNA sequence, it can effectively inhibit the mRNA

Further studies revealed that the long double-stranded interfering RNA was processed into short dsRNA (siRNA) of 21-23 nucleotides double stranded region with 3’ single stranded and Dicer ribonuclease is required for the processing

A Nobel Prize was awarded to Andrew Fire and Craig C. Mello on discovering RNAi in nematode worm

Reading List (IV): (i) RNA interference; (ii) Ribo-genome: the big world of small RNAs; (iii) Nobel

lecture on RNAi by Mello; (iv) Nobel lecture on RNAi by Fire.

Model for mRNA Translational Repression and RNA Interference Mediated by the RNA-Induced

Interference Complex (RISC)

• The double-stranded siRNA and miRNA are further processed into a multi-protein complex containing only one strand of RNA

• This RNA-induced silencing complex (RISC) cleaves target RNAs that is precisely complementary to their corresponding single-stranded siRNAs. These complexes also appear to function in the inhibition of translation by miRNA

miRNA Processing

• This diagram shows the processing of miR-1-1 miRNA

• Pre-miRNA is transcribed by RNA polymerase II

• Nuclear double-stranded specific endonuclease “Drosha” with its partner double-stranded RNA binding protein “DGCR8” make the initial cleaves in the pri-miRNA to generate a 70-nucleotide pre-miRNA

• The 70-nucleotide pre-miRNA is transported to cytoplasm by nuclear exporter

• In the cytoplasm, the 70-nucleotide pre-miRNA is processed by Dicer to form miR-1-1 miRNA

• One of the strands of miRNA is incorporated into an RISC complex with Argonaute protein

• The binding of several RISC complexes to an mRNA inhibits translation initiation

• Binding of RISC complexes cause the bound mRNPs to associate with dense cytoplasmic domains many times the size of a ribosome called cytoplasmic RNA-processing bodies, P-bodies, the site of RNA degradation

• About 1000 different human miRNAs have been observed, many of them expressed only in specific cell types

• miR-133, a specific miRNA is induced when myoblasts differentiate into muscle cells. miR-113 suppresses the translation of PTB, a regulatory splicing factor that functions similar to Sxl in Drosophila

• Knocking out of “Dicer” gene eliminates the generation of miRNA in mammals. In mouse knocking out Dicer gene will result in early embryonic death. If it is in limb primordia, it will result in abnormal limb development

Base Pairing with Target RNAs Distinguishes miRNA and siRNA

miRNA Function in Limb Development

Mouse embryos

Methods of Generating RNAi

Cytoplasmic Polyadenylation Promotes Translation in Some mRNAs

• 5’ and 3’ untranslated sequence in the mRNA could also contain regulatory functions

• In immature oocytes, many mRNA containing U rich and short poly (A) are inactive for translation. These mRNAs have to be polyadenylated in the cytoplasm before they can be activated for translation.

•The model used to explain the activation of these mRNAs is presented above.•CPE: cytoplasmic polyadenylation element; CPEB: CPE-binding protein•PABPI: poly (A) binding protein I; CPSF: cleavage & polyadenylation specificity factor; PAP: poly (A) polymerase

Pathways of Degradation of Eukaryotic mRNA

• The concentrations of an mRNA is a function of its synthesis and degradation

• Steady state level of mRNA: can be determined by extracting mRNA from a tissue and determine the level of the mRNA by various methods

• Bacterial mRNA is very labile, while mRNA from multi-cellular organisms is very stable

• Cytoplasmic mRNA is degraded by any one of the mechanisms listed above

Protein Synthesis Can Be Globally Regulated

• Synthesis of cellular proteins can be regulated via post-translational modification of initiation factors or ribosomal proteins via phosphorylation

• TOR Pathway: Rapamycin is an antibiotics produced by Streptomyces bacteria

which is able to suppress immune response in organ transplantation patients.

TOR (target of rapamycin) was identified in yeast by isolating yeast mutants resistant rapamycin inhibition of cell growth. TOR is a large protein kinase (2400 a.a.) that regulate several cellular processes in yeast cells in response to nutritional status. Metazoan TOR (mTOR) has also been isolated. It works similar to that of yeast TOR

• The pathway of mTOR is summarized in next slide Active mTOR stimulates the overall protein synthesis by

phosphorylating two proteins that regulate translation directlymTOR also activates transcription factors that control expression of ribosomal components, tRNAs, and translation factors, further activating cell growth with developmental programs as well as nutritional status

mTOR Pathway

• Active mTOR: mTOR complexes with Rheb-GTP

• Inactive mTOR: mTOR complexes with Rheb-GDP

• TCS1/TCS2-Rheb-GAP is phosphrylated by AMP-kinase

• Rheb-GAP: Rheb-GTPase activating protein which can be activated by AMPK

• TCS proteins were identified from human disease, tuberous sclerosis complex, forming benign tumors

mTOR Pathway (Continued)

The first step of translation of eukaryotic mRNA is binding of the eIF4 initiation complex to the 5’ cap via eIF4E cap-binding submit. The concentration of active eIF4E is regulated by a small family of homologous eIF4E-binding proteins (4E-BPs) that inhibits the interaction of eIF4E with mRNA 5’-cap

When 4E-BPs are phosphorylated by mTOR, 4E-BPs release eIF4E and resulted in stimulation of translation initiation.

mTOR also phosphorylates and activates another protein kinase that phosphorylates small ribosomal submit protein S6 (S6K), leading to further increase in the rate of protein synthesis

mTOR stimulates the translation of mRNAs with a string of pyrimidine in the 5’ untranslated region (TOP mRNAs for tract of oligopyrimidine) that encode ribosomal proteins and translation elongation factors

mTOR also activates RNA polymerase I transcription factor, TIF1A, activates transcription by RNA polymerase III, and activates two polymerase II activators that stimulate ribosomal genes

mTOR Pathway (Continued)

mTOR stimulates processing of the rRNA precursor mTOR activity is regulated by a monomeric small G protein in

the Ras protein family called Rheb. Rheb-GTP activates mTOR kinase. Rheb is in turn regulated by TSC1 and TSC2 heterodimer by hydrolyzing GTP to GDP

mTOR activity is also regulated in response to nutritional status. When energy from nutrients is not sufficient for cell growth, the resulting fall of ATP/AMP ratio is detected by AMP-kinase and phosphorylates TSC1/TSC2 to stimulate Rheb-GAP activity and leading to inhibition of mTOR kinase activity

In addition to regulating the global rate of cellular protein synthesis and production of ribosome, tRNAs, and translation factors, mTOR also regulates macroautophagy (degradation of cellular components).

eIF2 Kinase

• eIF2 kinases also regulate global rate of protein synthesis• eIF2 is a trimeric G protein existing in eIF2-GTP or eIF2-GDP

form, which can bind to charged initiator tRNA and in association with small submit of ribosome

• Phosphorylation of eIF2 -submit at a specific serine moiety resulted tight binding of eIF2 to GTP leading to failure of converting GTP to GDP, leading to inhibition of protein synthesis

• Cellular stress will lead to the same results• Human cells contain 4 eIF2 kinases that phosphorylate the

same inhibitory eIF2 -serine. Each of these is regulated by a different type of cellular stress

Iron-Dependent Regulation of mRNA Translation and Degradation

• Sequence –specific RNA-binding proteins control specific mRNA translation

• Transferrin binds ingested iron in order to transfer into cells

• Iron-transferrin complex binds to transferrin receptor residing on the cell membrane and the complex is transported into the cell by receptor-mediated endocytosis

• At low intracellular concentrations of iron, the IRE-BP (iron response element-binding protein) is active which can bind the stem loop of the AU-rich region and thus preventing the transferrin mRNA been degraded

• At high concentration of iron, IRE-BP is inactive and fails to bind to the stem loop of the AU-rich region of transferrin mRNA, thus no translation of ferritin mRNA

• IRE-BP also regulate TfR (transferrin receptor) mRNA by a similar mechanism

Nonsense-Mediated Decay and Other mRNA Surveillance

• There are several mechanism preventing improperly processed mRNA been translated into protein products

• mRNA surveillance is one of the mechanism to remove improperly spliced mRNA in the nulceus because the improperly spliced pre-mRNA is not transported out of the nucleus and is degraded in the nucleus

• Nonsense-mediated decay of mRNA: Degrades mRNA with one or more exons have been skipped. Exon-junction complex may be responsible for initiating nonsense-mediated decay of mRNA

Localization of mRNAs Permits Production of Proteins at Specific Regions within the Cytoplasm (I)

• For functional need some of the cellular proteins are localized in specific locations in the cell

• This is achieved by: transport the synthesized mRNA to the desired location or by distribution of mRNA in the desired location

• A well-documented example of mRNA localization occurs in mammalian myoblasts (muscle precursor cells). -actin mRNA is accumulated in the lamellipodia of the myoblasts

• When myoblasts fuse into myotube, expression of -actin mRNA is repressed and expression of -actin is seen in the perinuclear region

Localization of mRNAs Permits Production of Proteins at Specific Regions within the Cytoplasm (II)

This picture shows localization of neuronal mRNA to synapses. Sensory neuron from sea slug. The motor neuron is visualized in blue and green color is GFP-VAMP protein is in the synapse. The red color is the in situ hybridization of the GFP-VAMP mRNA also shown in the synapse

Processing of Ribosomal RNA Precursor

• Approximately 80% of the total cellular RNA is rRNA and 15% tRNA

• Pre-rRNA genes are similar in all eukaryotes and function as nucleolar organizers

• Pre-rRNA is encoded by a single-type of pre-rRNA transcription unit transcribed by RNA polymerase I

• Pre-rRNA is 45 S which is processed into 28 S, 18 S and 5.8 S rRNAs

• There are several common properties among rRNA gene from different species of eukaryotes: Pre-rRNA genes are arranged in tandem arrays separated by non-

transcribed spacer regions ranging from 2.0-30 kb Genomic regions corresponding to the three mature rRNAs are arranged

in the same 5’ to 3’ order: 18S, 5.8S and 28S The transcribed regions are processed and degraded rapidly

EM of Pre-rRNA Transcription Units from the Nucleolus of a Frog Oocytes

General Structure of Eukaryotic Pre-rRNA Transcription Unit

Processing of Pre-rRNA and Assembly of

Ribosomes in Higher Eukaryotes

• Small nucleolar RNAs (snoRNAs) assist the processing of pre-rRNA and assembling of ribosome submits

• Pre-rRNA binds to proteins to form ribosomal ribonucleoprotein particles (pre-rRNPs)

• Small Nucleolar RNAs (snoRNAs) can form snoRNP by hybridizing to pre-rRNA

• 5S RNA is transcribed from 5S RNA gene by Pol III

Pre-tRNAs Undergo Cleavage, Base Modification and Sometimes Protein-Catalized Splicing

• A 5’ sequence of variable length is precessed out from the pre-tRNA by ribonuclease P (RNase P)

• About 10% of the bases in tRNA is modified during processing

• Three types of modifications: (i) replacing U at the 3’ end with CCA; (ii) addition of methyl and isomethyl groups to the heterocyclin ring of purine base and methylation of the 2’-OH group in the ribose of any residue; (iii) conversion of specific U to dihydro-U, Pseudo-U or ribothymidine

• Splicing of intron in pre-tRNA is different from splicing of type I and type II introns in pre-rRNA and splicing introns in pre-mRNA in the following three ways: Splicing of pre-tRNA is catalyzed by proteins, not by RNAs A pre-tRNA intron is excised in one step that entails

simultaneous cleavage at both ends of the intron Hydrolysis of GTP and ATP is required to join the two tRNA

halves generated by cleavage on their side of the intron

• Export of tRNA to cytoplasm require the mediation of exportin-t

• Generally speaking, tRNAs in the cytoplasm rarely stay alone without in association with protein factors such as aminoacyl synthetase, elongation factors and ribosomes

Conclusion of Regulation of Gene Expression

• Regulation at transcriptional level: Regulation of initiation of transcription

Chromatin-mediated transcriptional control Activators and repressors interaction with transcription complex

• Regulation at post-transcriptional level in the nucleus: Regulation of alternative splicing leading to production of

multiple isoforms of proteins Regulation of transport of mRNA into cytoplasm

• Regulation at post-translational level in cytoplasm Micro RNAs RNA intereference (RNAi or siRNA) Cytoplasmic polyadenylation mRNA degradation Localization of mRNA in the cytoplasm

Assigned Reading

1.hnRNP complex2.Heterogenous ribonuclear particles3.R Loop Mapping4.Nobel Lecture by Richard Sharp5.Mapping of viral RNA with viral DNA6.Cech’s Nobel Prize lecture7.Hammerhead ribozyme8.Distribution of Ca++-activated K+ chennel isoforms along the tonotopic gradient 9.Micro RNA 10.Micro RNA controlling cell division – differentiation and cell death