RNA Processing or post transcriptional modifications

• Primary transcripts is linear, RNA copy of a transcriptional unit.

• Primary transcripts of both prokaryotic and eukaryotic tRNA and rRNA are modified by cleavage of the original transcripts by ribonucleases.

• Prokaryotic mRNA is generally identical to itsprimary transcript, whereas eukaryotic mRNA is extensivey modified.

A. rRNA

• r- RNA of both prokaryotic and eukaryotic cells aresynthesized from long precursor molecule called preribosomal RNA.

• From single precursor molecule 28S, 18S and 5.8S rRNA are produced in eukaryotes and 23S, 16S and 5S rRNA are produced in prokaryotes.

• The nucleases that cleave and trim these precursors of rRNA and tRNA are highly precise.

Primary Transcript

B. tRNA

• tRNA precursors are converted into mature tRNAs by a series of alterations:

• cleavage of a 5 leader sequence• splicing to remove an intron• replacement of the 3 -terminal UU by CCA and • modification of several bases

Transfer RNA Precursor ProcessingThe conversion of a yeast tRNA precursor into a mature tRNArequires the removal of a 14-nucleotide intron (yellow), the cleavage of a 5 leader (green), and the removal of UU andthe attachment of CCA at the 3 end (red). In addition, several bases are modified.

C. Eukaryotic mRNA

• most extensively modified transcription product is the product of RNA polymerase II:

• The immediate product of an RNA polymerase is sometimes referred to as pre-mRNA.

• Most pre-mRNA molecules are spliced to remove the introns.

• both the 5 and the 3 ends are modified

1. 5’ Capping

• 5 triphosphate end of the nascent RNA chain is immediately modified.

• First, a phosphate is released by hydrolysis.• The diphosphate 5 end then attacks the a-• phosphorus atom of GTP to form a very unusual

5 -5 triphosphate linkage. This distinctive terminus is called a cap.

• The N-7 nitrogen of the terminal guanine is then methylated by S-adenosylmethionine.

• Transfer RNA and ribosomal RNA molecules, in contrast with messenger RNAs and small RNAs that participate in splicing, do not have caps.

• Caps contribute to the stability of mRNAs by protecting their 5 ends from phosphatases and nucleases.

• In addition, caps enhance the translation of mRNA by eukaryotic proteinsynthesizing systems.

Capping the 5’ End.

2. Poly-A tail

• Pre-mRNA is also modified at the 3 end.• Most eukaryotic mRNAs contain a polyadenylate,

poly(A), tail at that end, added after transcription has ended.

• DNA does not encode this poly(A) tail.• Eukaryotic primary transcripts are cleaved by a specific

endonuclease that recognizes the sequence AAUAAA.• After cleavage by the endonuclease, a poly(A)

polymerase adds about 250 adenylate residues to the 3 end of the transcript; ATP is the donor in this reaction.

• The role of the poly(A) tail is still not firmly established despite much effort. However, evidence that it enhances translation efficiency and the stability of mRNA is accumulating.

• Blocking the synthesis of the poly(A) tail by exposure to 3 -deoxyadenosine (cordycepin) does not interfere with the synthesis of the primary transcript.

• Messenger RNA devoid of a poly(A) tail can be transported out of the nucleus. However, an mRNA molecule devoid of a poly(A) tail is usually a much less effective template for protein synthesis than is one with a poly(A) tail.

Polyadenylation of a Primary TranscriptA specific endonuclease cleaves the RNA downstream ofAAUAAA. Poly(A) polymerase then adds about 250 adenylate residues

3. Removal of introns

• Most genes in higher eukaryotes are composed of exons and introns.

• The introns must be excised and the exons linked to form the final mRNA in a process called splicing.

• This splicing must be exquisitely sensitive: a one-nucleotide slippage in a splice point would shift the reading frame on the 3 side of the splice to give an entirely different amino acid sequence. Thus, the correct splice site must be clearly marked.

• The base sequences of thousands of intron- exon junctions within RNA transcripts are known.

• In eukaryotes from yeast to mammals, these sequences have a common structural motif: the base sequence of an intron begins with GU and ends with AG.

• The consensus sequence at the 5 splice in vertebrates is AGGUAAGU.

• At the 3 end of an intron, the consensus sequence is a stretch of 10 pyrimidines (U or C), followed by any base and then by C, and ending with the invariant AG.

• Introns also have an important internal site located between 20 and 50 nucleotides upstream of the 3 splice site; it is called the branch site

• In yeast, the branch site sequence is nearly always UACUAAC, whereas in mammals a variety of sequences are found.

• The splicing of nascent mRNA molecules is a complicated process.

• It requires the cooperation of several small RNAs and proteins that form a large complex called a spliceosome

• Splicing begins with the cleavage of the phosphodiester bond between the upstream exon (exon 1) and the 5 end of the Intron. The attacking group in this reaction is the 2 -hydroxyl group of an adenylate residue in the branch site. A 2 ,5 -phosphodiester bond is formed between this A residue and the 5 terminal phosphate of the intron. Hence a branch is generated at this site, and a lariat intermediate is formed.

• The 3 -OH terminus of exon 1 then attacks the phosphodiester bond between the intron and exon 2. Exons 1 and 2 become joined, and the intron is released in lariat form.

Splice SitesConsensus sequences for the 5 splice site and the 3 splice site are shown. Py stands forpyrimidine.

Splicing Mechanism Used for mRNA Precursors.

Small Nuclear RNAs in Spliceosomes Catalyze the Splicing of mRNA Precursors

• The nucleus contains many types of small RNA molecules with fewer than 300 nucleotides, referred to as snRNAs (small nuclear RNAs). A few of them designated U1, U2, U4, U5, and U6 are essential for splicing mRNA precursors. The

• These RNA molecules are associated with specific proteins to form complexes termed snRNPs (small nuclear ribonucleoprotein particles); investigators often speak of them as "snurps."

• Spliceosomes are large (60S), dynamic assemblies composed of snRNPs, other proteins called splicing factors, and the mRNA precursors being processed

Spliceosome AssemblyU1 (blue) binds the 5 splice site and U2 (red) to the branch point. A preformedU4-U5-U6 complex then joins the assembly to form the complete spliceosome

Alternative splicing

• It is a widespread mechanism for generating protein diversity.

• The differential inclusion of exons into a mature RNA, alternative splicing may be regulated to produce distinct forms of a protein for specific tissues or developmental stages.

• recent estimates suggest that the RNA products of 30% of human genes are alternatively spliced.

Alternative Splicing PatternA pre-mRNA with multiple exons is sometimes spliced in different ways.Here, with two alternative exons (exons 2A and 2B) present, the mRNA can be produced with neither, either, or bothexons included. More complex alternative splicing patterns also are possible