RNA Processing

4 key unique steps eukaryotic mRNA’s undergo

1. Addition of 5’ Cap

2. Addition of Poly (A) tail

3. Splicing

4. Nuclear export

what is Pre-mRNA?

the immediate product of transcription

• the direct readout basepair of what the DNA is, prior to anything being done, full of introns

what is mRNA

fully processed transcript

• removal of introns and ready to do translation, you have the methyl cap of 5’ end and the poly(A) tail on 3’

What is the 5’ cap?

a modification made to the 5’ shortly after transcription begins

only Pol II transcripts are capped

What is the 5’ Cap for?

• protects end of transcript from degradation by 5’ to 3’ exonuclease (chews up RNA if not protected)

  • any loose RNA that can infected by viral viruses

• acts as a handle used by many proteins that bind to transcripts

• required for traditional translation

What is the capping molecule?

7-Methylguanosine

• a GTP added backwards onto the 5’ phosphate of the transcript, then methylated at the 7 position

How is the 5’ cap added?

by an enzyme called guanylytransferase aka the
“capping enzyme”

How does the capping enzyme cap?

• guanylyltransferase binds to the CTD of Pol II

• as the mRNA leaves Pol II the 5’ end sticks to the CTD, allowing the enzyme to cap it

what happens after the addition of the capping enzyme?

it falls off and a protien called CBC (cap binding complex) binds to the cap

CBC and the 5’ end of the transcript remain on the CTD

what is polyadenylation “poly (A) tail”

a long tail of A’s (adenosines) added to the 3’ end of every mRNA

What is the Poly A tail for?

• protects 3’ end from degradation

• helps recruit to ribosomes for translation

• imports for mutations that occur, helps defend

How is the Poly A tail added?

Step 1:

• Pol II transcribes past termination sequence or the Poly A addition site (AAUAAA) where CPSF (cleavage-poly adenylation cleavage factors) attach to the CTD

• it then cuts the mRNA downstream of the AAUAAA before GU region

Step 2:

• PAP (polyadenylate polymerase) begins adding poly A binding proteins (PABP) attaches to the tail, protecting it

What is splicing?

the removal of extra sequences that don’t encode for amino acids

what are exons?

parts of the mRNA that aren’t present in the final transcript

EXPRESESS PROTEIN

what are introns?

regions in between exons that don’t encode amino acids and are the ones being removed

INTERRUPTS CODING SEQUENCE

why are introns important to have?

they can contain sequences involving gene regulation

what is alternative splicing (isoforms)

they are partially of entirely skipped exons

• these exons are NOT re-arranged in splicing, they still remain in numerical order

What machinery does the splicing?

spliceosomes

what is splicing mechanism characteristics?

• snRNPs facilitate each step of the reaction

• snRNPS are recruited essentially in numerical order

• RNA sequences on the same snRNAs interact with sequences on the 5’ and 3’ splice sites as well as the branch point

• the RNA of the snRNPs catalyze the splicing reaction

what 3 things occur during transcription?

capping

polyadenylation

splicing

all involve the machinery binding to the CTD

the 5’ end of transcript remains tether to CTD the whole time

after processing what must happen to mRNAs?

must be exported to the cytoplasm to be translated (where the ribosomes are)

Can RNA’s be re-imported back into the nucleus?

True

what must happen to mRNAs after translation?

the must be degraded so they can be recycled

what are non-protein coding RNA’s?

exportins and importins shuttle RNA out of or into the nucleus

both in Pol I and Pol II

What are Exports?

• Ran-GTP binds to exporting and RNA

• complex exits nucleus

• Ran-GTP hydrolyzed to Ran-GDP, releasing RNA and exportin

  • it is then shuttled back into the nucleus to begin again

what are Imports?

• Ran-GDP binds to importing and RNA

• complex re-enters nucleus

• Ran-GDP converts back to Ran-GTP, and releases RNA

what is mRNA export

• uses TREX instead of exportin/Ran-GTP

• TREX recruited to Pol II

how does splicing help target mRNA’s for export?

• creates exon junction complexes (EJCs) at borders between exons

• export machinery uses EJCs to help target spliced transcripts

Why use EJCs?

they distinguish pre-mRNA from mRNA

What removes the 5’ cap and poly (A) tail

exoribonucleases

what are P bodies?

unclear function

storage place for mRNAs, thought to be involved in RNA degradation but also in storage prior to translation