RNA ProcessingRNA Processing

• Capping

• Polyadenylation

• Introns vs exons

• Splicing

• Genomic vs cDNA

• Ribosomal RNA processing

• t-RNA processing

Prokaryotes vs Eukaryotes

PRO: All three classes of RNA are synthesized by onepolymerase.EU: There are 3 major RNA polymerases. Pol 1 synthesizes rRNA; Pol 2 synthesizes mRNA; Pol 3synthesizes tRNA.

PRO: mRNA undergoes hardly any posttranscriptionalprocessing. It is translated as it is synthesized.EU: mRNA is capped, polyadenylated, spliced

PRO: mRNA contains no introns

EU: mRNA contains intervening sequences (introns) that are removed during processing

mRNA CappingRULE: Capping the 5’ endof mRNAs serves 2 purposes. First, the cap protects the mRNA from 5’-exonuclease activity.

Second, the cap interacts witheIF-2, a translation initiation factor required to position the mRNA on the ribosome

CH3

CH3

CH3

Added Guanine

5’-5’ triphosphateGppp + pppApNpNp…

GpppApNpNp… + pp + p

Polyadenylation

In eukaryotes, mRNAis polyadenylated byan enzyme systemthat cuts the RNA 30 bsdownstream from anAAUAAA, thenadds A to the 3’ endat the cleavage site

In eukaryotes, mRNAis polyadenylated byan enzyme systemthat cuts the RNA 30 bsdownstream from anAAUAAA, thenadds A to the 3’ endat the cleavage site

Thus, poly A is notcoded in the DNA, butis added after transcription

DialogueQ: How should one picture a typical mammalian gene?

A: Mammalian genes have both introns and exons.Only the exons encode information that will appear in a protein.Q: What are introns?

A: Introns appear in unprocessed mRNA. The term isa shortened version of the words “intervening sequences”

Q: How do exons differ from introns?

A: One could say that the typical mammalian gene has7 or 8 exons in a length of about 15 kb. The exons are short, (100-200 bp) whereas introns are large (>1000 bp)

Q: What happens to introns?

A: During nuclear processing, the introns are spliced out and exons are joined together in a linear continuumQ: How is this accomplished?

A: Cells have mechanism that recognize introns. The most common is a spliceosome that recognizes the boundaries of intron-exon junctions and knows were to cleave and splice

Q: What is involved in the recognition?

A: Small RNA molecules that work with spliceosomes, The RNA hybridizes to the residues at the splice junctions and tells the enzyme where to cut.

Stages in the Life of a Typical mRNA

DNA

Transcription via RNA Pol II

Primary Transcript

Capping and polyadenylation

Capped-polyadenylated mRNA

Splicing

Mature mRNA

Introns form loops that help with their excision and splicing

Intron I is generallyquite large in mammalian mRNAs

Chick Ovalbumin

1,872 nucleotides (24.3%) represented as exons

What can you tell me about splicing?

Dialogue on mRNA Splicing

Ans: Splicing occurs in two steps. First the junction at the5’-end of the intron is broken. This is called the 5’-splice site

Then what happens?

Ans: The -OH on the 3’ end of the liberated exon becomes a nucleophile and attacks the 3’-splice site of the intron. This isthe second step. The two exons are now joined.

What happens to the intron?

Ans: The intron is set free. Because a 2’-OH on an adenosine caused the initial cleavage, there is a loop in theintron (called a lariat)

Does this happen automatically

Ans: Sometimes. Some RNAs are capable of self-splicing.Most of the time the splicing occurs through a large complexcalled a spliceosome.

What is a spliceosome?

Ans: A spliceosome is a giant 50-60S particle composed of splicing proteins, pre-mRNAs and small nuclear RNA proteins or “SNURPS”.

What do the snRNPs do?

Ans: The 5’-end of some of the snRNPs complements bases at the splice junctions. The snRNPs probably locate theexon-intron boundaries which tend to be constant for alleukaryote mRNAs. There are about 6 of them U1-U6. Notall functions are known.

OH2’

pApA pGpU pApG pGp

C AU1-snRNP

pApA pGpU pApG

O

pGp

OH

AdenosineExon 1 Exon 2

pApA pGp

Spliced exons

Exon 1 Exon 2

Intron

pGpU pApG

O

Intron with lariat

-OH3’

Ribosomal RNAQ: Is ribosomal RNA processed the same way as mRNA?A: No

Q: How is it different?A: In bacteria, r-RNA is not spliced, it is only cut. All processing is done with a special class of RNAases

Q: What about eukaryotes?

A: Eukaryotes employ basically the same mechanism, but they also can engage in self-splicing

Q: What is self-splicing?

A: Self-splicing implies that a pre-rRNA can carry out its own splicing without the need of a spliceosome.

Q: Does this mean the RNA is acting as its own nuclease?A: Yes. It is called a ribozyme in recognition of itscatalytic activity.

Q: How does self-splicing work?A: In self-splicing 3’-OH group on the donor exon isprimed by an attack by GMP, GDP, or GTP.

Q: Then what?A: As before, the freed -OH attacks the phosphate at the3’ end and forms a new linkages that joins to two segments

Secondary processing

III III III IIIP

PF

PF E

Ribosomal RNA Primary transcript

Primary processing

1700 2920200150 300

5’ 3’

RNase:

M16 M16 M23 M23 M5D D

Pre 16S rRNA Pre 23S rRNA

Pre-5S rRNA

5’ 3’

RNase:

16S rRNA 23S rRNA

1541 2904 120

tRNA(s) tRNA(s)

5S rRNA

5’ 3’Numberof bases

Bacteria rRNA

16S 23S 5S4S 4S

Stages in the Life of a Eukaryotic Ribosomal RNA

DNA (300 randomly repeated copies of rRNA genes in the genomethat are transcribed via RNA pol I and processedin the nucleolus)

Primary Transcript 5’ 3’18S 5.8S 28S

45S RNA

Methylation at 110 sites

Methylated Transcript

RNase III,RNase P tRNA and 5S sequences

are not part of the 45S transcript

spacers

Heavily methylated

Self-splicing pre-rRNA

Any guanine nucleotide (GMP, GDP,GTP) sets it off

Processing t-RNA

CutSpliced out

Note: mature t-RNA has the highest numberof odd bases

Amino acidattachment site

Anticodon loop

pseudouridine

isopentenyladenosine

2’-methylguanosine

dihydrouridine

See p345in Strategies

methyladenosine