RNA Processing

Bacterial RNA Processing

Eukaryotic RNA Processing

Transcription and Translation are simultaneous and occur in the cytoplasm bc no nucleus.

No capping

No splicing

Splicing = removal of introns

No PolyA Tails

Cistronic-ness

Polycistronic = when one mRNA makes multiple gene products

Monocistronic = when one mRNA makes one product

Remember there can be multiple open reading frames/exons but if they only make one product, they are still monocistronic

only polycistronic if multiple gene products are made

ORF1 + ORF2 + ... = an operon if they are all on the same mRNA

Why is it good to have multiple products on one mRNA?

Keep shit together that works together

turn on/off expression only once per unit of working parts

increased efficiency

helpful bc mRNA has a very short half life (minutes)

Parts of mRNA

Open reading frame (ORF)

start codon is at beginning of first ORF

5' untranslated region (UTR)

3' UTR

contains information on protein folding and ribosome loading

mRNA has to exported out of the nucleus before translation.

ribosomes are in the cytoplasm outside of the nucleus

exportation allows more regulation

Parts of mRNA

25.9% introns

1.5% protein coding

Processing takes place in the CTD tail of RNAP (aka still in the nucleus) via the following modifiers (in order). Processing is co-transcriptional.

promoter escape

DNA repair

capping

splicing = removal of introns from pre-mRNA

polyA tail addition (polyadenylating)

the 5' cap is called 7-methyl-G

functions of 7-methyl-G

signal for nucleus export

assists in ribosome loading

promotes mRNA circularization

the cap sticks to the polyA tail

protection from RNAase because mRNA is now unrecognizable to the enzyme

3 enzymatic steps all occur at C-terminus

  1. RNA triphosphate removes gamma phosphate from the 5' end
  1. guanylyltransferase adds GMP Moiety to the beta phosphate on the 5' end of another mRNA

leads to condensation reaction called 5' to 5' joining

  1. methyltransferase adds the 7-methyl-G cap to mRNA

DNA codes for polyA sequence and transcribes it onto RNA

sequence = 50-250 A's all in a row

steps of polyA tail addition

  1. Pol II transcribes the polyA tail
  1. CTD associated endonuclease binds to mRNA

cuts 20-40 nucleotides downstream

  1. 3' OH is freed
  1. 3' OH is bound by polyA polymerase (PAP)
  1. PAP adds the polyA tail
  1. PolyA binding protein (PABP) coats the polyA tail and protects the mRNA

increases mRNA half-life

increases mRNA half-life

Quick Facts

average human gene has 8 introns

average exon length = 150bp

average intron length = 3000bp

muscles have the most introns

Largest pre-mRNA is 2,400,000 nt long

pre-mRNA = mRNA before splicing

transcription rate = 50 nt/sec

bigger mRNA's tend to have a longer half-life and lower abundance

pre-mRNA binds to DNA's non-introns and the introns stick out in loops to be cleaved later

Splicing factors are on the CTD tail and this process is co-transcriptional

Splicing vs Alternative Splicing

Splicing includes all of the exons

Alternative Splicing excludes one or more exons to make a totally different gene products

same gene can code for different products based on splicing behavior

variance in splicing pattern leads to different products (called isoforms)

No introns

Makes operons

UTR's do not get translated into amino acids

determines speed of degradation

there's a triphosphate here

Typically, eukaryotic mRNA is monocistronic

occurs during transcription

image

promotes circularization

Functions of Introns

coding and can sometimes become exons

code for functional RNA

allows alternative splicing

Variation is based on which poly A site is acknowledged

Order of exons is NEVER scrambled

Types of splicing

Group 1 Introns (NOT Covered)

Group 2 Introns (NOT Covered)

Nuclear pre-mRNA Splicing

Most common

Mechanism

1) Branch point A at 2' OH attacks phosphate at 5' splice site

this makes a covalent bond

forms lariat structure

transesterification reaction 1 catalyzed by U2, U5, and U6

2) Activated 5' splice site attacked 3' splice site

3) Intron lariat is released and degraded

4) exons are spliced together

Recognition sites for nuclear pre-mRNA splicing

5' splice site with a GU

3' splice site with an AG

Branch point A with a 2' OH

Enzymes = snRNP's

enzymes = nonspontaneous rxn

100-300 nt RNA + proteins = snRNP

Roles of snRNP's

recognition of 5' splice site and branchpoint A

bring 5' splice site and 2'OH of branchpoint A together

bring 3'OH upstream of the exon's 5' splice site so it's close to the phosphate of the 3' splice site

Catalyzes transesterification reactions

Order of rxns carried out by snRNP's

1) U1 binds at 5' splice site of GU of intron

2) U2 binds to branchpoint causing A to bulge out

3) U1 + U2 complex brings 2'OH to the 5' spice site

4) U4 + U5 + U6 complex joins the U1 + U2 complex

5) proximity of the 2' OH and 5' splice site leads to transesterification rxn 1 catalyzed by U2, U5, and U6

U1 and U4 are displaced

U2 is at branchpoint A

6) transesterification rxn 2 is catalyzed by U2, U5, and U6

exons spliced togetehr

intron lariat is released

Very energy intensive

Activates 5' splice site

transesterification rxn 2 catalyzed by U2, U5, and U6

free 3'OH at 5' splice site attacks phosphate of next exon at 3' splice site

sticking to the cap

Most regulation occurs during transcription

interactions can be any combination of RNA and protein interactions (protein::protein, RNA::RNA, or RNA::protein)