RNA Processing

M.Prasad NaiduMSc Medical Biochemistry, Ph.D,.

Overview of the Eukaryotic mRNA Processing

Eukaryotic cells process the RNA in the nucleus before it is moved to the cytoplasm for protein synthesis

The RNA that is the direct copy of the DNA is the primary transcript

Two methods are used to process primary transcripts to increase the stability of mRNA for its export to the cytoplasmRNA cappingPolyadenylation

RNA capping happens at the 5’ end of the

RNA, usually adds a methylgaunosine shortly

after RNA polymerase makes the 5’ end of the

primary transcript

Splicing of introns removes the intervening

sequences in RNA

Polyadenylation modifies the 3’ end of the

primary transcript by the addition of a string

of As

Over all Processes

Modified guanine nucleotideadded to the 5 end

Protein-coding segment

3 UTRStop codonStart codon

5 Cap 5 UTR

AAUAAA

TRANSCRIPTION

RNA PROCESSING

DNA

Pre-mRNA

mRNA

TRANSLATION

Ribosome

Polypeptide

G P P P

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a) 5’ Capping of Transcript

Modified GTP is added, backwards, on the 5’ end

After about 30 nt are added, 5’-P is almost immediately modified

A phosphate (terminal) is released by hydrolysisThe diphosphate 5’ end then attacks the alfa

phosphate of GTP to form a very unusual 5’-5’ triphosphate linkage – this is called condensation

This highly distinctive terminus is called a capThe N-7 nitrogen of the terminal G is then methylated

by S-adenosyl methionine to form cap0

Uses of CappingCaps are important for subsequent splicing

reactions

They also 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 protein-synthesizing systems

Note: tRNA and rRNA molecules do not have caps

b) Poly-AdenylationMost Eukaryotic mRNAs contain poly A tailPoly A tail is not encoded by DNASome mRNAs contain an internal AAUAAA (AAU

= Asn, AAA = Lys). This highly conserved sequence is only a part of the cleavage signal, but its context is also important

The cleavage site is 11 to 30 nt away from the AAUAAA site on the 3’ side

After the cleavage by an endonuclease, 50 to 250 A residues are added by Poly adenylate polymerase

50 to 250 adenine nucleotidesadded to the 3 end

Protein-coding segment Polyadenylation signal

Poly-A tail3 UTRStop codonStart codon

5 Cap 5 UTR

AAUAAA AAA…AAA

TRANSCRIPTION

RNA PROCESSING

DNA

Pre-mRNA

mRNA

TRANSLATION

Ribosome

Polypeptide

G P P P

53

Cleavage site

Mutating the cleavage sequence in the parent DNA will result in mRNA that is not polyadenylated and not exported to the cytoplasm – instead it is rapidly degraded

A second downstream signal that is a G/U rich sequence is required for efficient cleavage and polyadenylation, and is located ca. 50 nucleotides from the site of cleavage.

The cleavage and polyadenylation specficity factor (CPSF), a large 4-subunit protein (ca. 360 kDa), forms an unstable complex with the AAUAAA sequence that is subsequently stabilized by the addition of at least 4 separate protein complexes that bind to the CPSF-RNA complex.

CstF: Cleavage stimulatory factor interacts with G/U rich sequence CFI: Cleavage factor I and CFII help stabilize protein-RNA complex PAP: Poly(A) polymearse binds to complex before cleavage occurs PABP: Polyadenylate-binding protein binds the Poly (A ) polymerase

Assembly of the cleavage/polyadenylation complex

Cleavage and polyadenylation Specificity Factor

Cleavage Stimualtory Factor

(PABP)

Cleavage site

(i)

(ii)

CPSF

PAP

(iii)

TRANSCRIPTION

RNA PROCESSING

DNA

Pre-mRNA

mRNA

TRANSLATION

Ribosome

Polypeptide

5 Cap

Exon Intron

1

5

30 31

Exon Intron

104 105 146

Exon 3

Poly-A tail

Poly-A tail

Introns cut out andexons spliced together

Codingsegment

5 Cap

1 1463 UTR5 UTR

Pre-mRNA

mRNA

c) Splicing out Introns

RNA splicing is responsible for the removal of the introns to create the mRNA

Introns contain sequences that act as clues for their removal

Carried out by assembly of small nuclear ribonucleoprotein particles (snRNPs) – Spliceosomes

Spliceosome ActivitysnRNPs come together and cut out the intron

and rejoin the ends of the RNAU1 snRNP attaches to GU of the 5’ intronU2 snRNP attaches to the branch siteU4, U5 and U6 snRNPs form a complex

bringing together both U1 and U2 snRNPsFirst the donor site is cut followed by 3’ splice

site cut

Intron is removed as a lariat – loop of RNA like a cowboy rope

(U1, U2, U4, U5 and U6)

Mechanism of Splicing

1. The branch-point A nucleotide in the intron sequence, located close to the 3’ splice site, attacks the 5’ splice site and cleaves it.

The cut 5’ end of the intron sequence becomes covalently linked to this A nucleotide

2. The 3’-OH end of the first exon sequence that was created in the first step adds to the beginning of the second exon sequence, cleaving the RNA molecule at the 3’ splice site, and the two exons are joined

Thomas Cech (1981)

Nobel prize in 1989

Exception: RIBOZYME

Self-splicing of Intron SequencesGroup I intron sequences bind a free G

nucleotide to a specific site to initiate splicingGroup II intron sequences use s specially

reactive A nucleotide in the intron sequence itself for the same purpose

Both are normally aided by proteins that speed up the reaction, but the reaction is mediated by the RNA in the intron sequence

The mechanism used by Group II intron sequences forms a lariat and resemble the activity of spliceosomes

Comparison

Alternative Splicing Patterns1, 2A, 3 1, 2B, 3

1, 2A, 2B, 3 1, 3

(Calcitonin-gene related protein)

Two predominant Poly(A) sites in Rats

Cell type specific RNA splicing

Processing of pre-rRNA transcripts

Benefits of SplicingAllows for genetic recombination

Link exons from different genes together to create a new mRNA

Also allows for one primary transcript to encode for multiple proteins by rearrangement of the exons

RNA Editing

How do mRNAs get to the cytosol?

Why do eukaryotes have DNA within a membrane bound compartment and prokaryotes do not?

Could eukaryotes function without it?

Correspondence between exons and protein domains

GeneDNA

Exon 1 Intron Exon 2 Intron Exon 3

Transcription

RNA processing

Translation

Domain 3

Domain 1

Domain 2

Polypeptide

Sequences removed are called Introns

Coding sequences flanking introns are called Exons

Exons are not removed and are in the mRNA

Intron removal is referred to as Splicing

Splicing is mediated by a particle: Spliceosome

A spliceosome is made of snRNA and protein

There are several snRNAs in a spliceosome, U1 to U6 Some introns have self-splicing sequences: Ribozymes

Conclusions