mRNA Processing

What do eukaryotes have that prokaryotes dont?

cistronic RNA, 5’ caps, poly-A tail.

Step 1 of mRNA processing

mRNa processing requires C-terminal domain of RNA polymerase II

Step 2 of mRNA processing

CTD contains repeats of heptapeptide YSPTSPS. S5 and S2 are differentially phosphorylated during initation and elongation

Step 3 of mRNA processing

Phosphorylation of CTD provides binding site fro factors involved in capping, splicing, and 3’ end formation

Step 4 of mRNA processing

These factors are transferred to the nascent RNA at the appropriate time

What is the structure of the 5’ cap?

a 7-methylguanosine (the cap) attached to the 5’ end of primary transcript via a 5’ to 5’ triphosphate bridge

Also the N on the 7-methylguanosine is methylated

Reaction 1 of 5’ capping

RNA 5’ phosphatase (RTP)

Reaction 2 of 5’ capping

Guanyl transferase (GT)

Reaction 3 of 5’ capping

Guanine 7-methyl transferase (MT)

Is Capping of pre-mRNA coupled with transcription?

Yes, as the capping enzymes are coupled with CTD.

The cap binds to the cap-binding complex, which facilitates further mRNA processing and export

Why is 5’ capping necessary?

Cap distinguishes mRNAs from other types of RNA molecules

mRNAs need a cap (and poly A tail) for export from nucleus

Necessary for translation in cytoplasm: translation initiation factors

cap stabalizes mRNA in cytoplasm

Exons

Expressed

Introns

Remain In the nucleus

What is the structure that introns are removed in?

They are called lariats!

Consensus sequences involved in splicing

It seems that AGGU is important for the donor portion

And that AGG is important for acceptor?

A just needs to be in the branch site so it can do its thing

What are the two types of splicing errors?

Exon skipping

Cryptic splice site selection

What is cryptic splcie site selection?

Nucleotide sequences of RNA that closely resemble true splicing signals and are sometiems mistakenly used by the spliceosome

SR protiens

Serine/arginine-rich protiens

they promote binding to the area of recognition and lead to splicing

HnRNP

They repress splicing

The first come first served model

The first things to be synthesized get to be spliced and dealt with first. Also works with the idea that RNA generation and splicing happens at the same time

Constitutive RNA splicing

Results in a single form of mature mRNA from a primary transcript

Alternative RNA splicing

Works with the idea that mRNA can be spliced a number of different ways

Factors that regulate this include splice site stregnth, Cis-regulatory sequences, avundance of trans-acting factors, and elongation rate

What do strong splice site lead to?

Usually lead to constitutive splicing

What do weak splice sites lead to?

Give rise to different mRNA isoforms

What are ESE or ESS?

Exonic splicing enhancer or silencer

What are ISE or ISS?

Intronic splicing enhancer or silencer

How does alternative splicing affect telomere maintenance?

By leaving out an exon, it can lead to the mRNA degredation and eventually telomere shortening

Beta thalassemia

Server anemia due to aberrant hemoglobin sythesis, caused by splice site mutations

CPSF

cleavage and polyadenylation spcificity factor

CstF

clevage stimulation factor

CF

clevage factor

Alternative polyadenylation

The presence of multiple polyadenylation sites, different choices on the primary transcript, present in >50% human genes

What can mRNA surveillance act as?

It can be a quality control mechanism, monitoring to see what mRNAs look really werid and can initiate their deletion

What does the Nuclear Pore Complex do?

Regulates what mRNAs make it into the cytoplasm, acting as a gate.

The exosome

a 3’ to 5’ RNA exonuclease

It will degrade aberrant mRNA but allow properly processed mRNA to be exported

Nonsense mediated decay

Eliminates mRNAs containing premature termination codons

Recognized by exon junction complex (EJC)

Recruits NMD factors to trigger aberrant mRNA decay, requires translation to trigger mRNA decay