RNA processing

Question 1

4 steps

Answer 1

5’cap
cleavage
polyadenylation
splicing

Question 1

5’cap

Answer 1

during pausing of transcription

• A 7’ methylguanylate cap is added to the 5’ terminal nucleotide through an unusual 5’- 5’ triphosphate linkage (on first ribonucleotide with GTP)
• In animal cells and in higher plants the 2’hydroxyl of the ribose group of the first base is methylated.
• In vertebrates the second nucleotide is also methylated.

CTD Ser 5 phosphorylation bind with capping enzyme

CTD ser2 phosphorylation

Question 2

cap function

Answer 2

Addition of the cap:
-protects the pre-mRNA from degradation
-facilitates nuclear export
-assists recognition by translation factors

Question 3

Ser-5 phosphorylation ensures

Answer 3

only RNAs transcribed by Pol II
(mostly mRNAs) are capped

Question 4

Serine 2 phosphorylation recruits additional proteins

Answer 4

Splicing factors
Polyadenylation factors
Export factors

Question 5

poly(A) signal

Answer 5

3‘ AAUAAA xxxxx G/U sequence
recognition and binding site

Question 6

cleavage and polyadenylation factors

Answer 6

CPSF, CStF, CFI, CFII

Question 7

formation of poly (A) tail

Answer 7

poly(A) polymerase (PAP) add~12 A residues
nuclear poly (A) binding protein
(PABPN1) which catalyzes the rapid addition of ~200 A residues.

Question 8

Are all mRNA transcripts polyadenylated?

Answer 8

All mRNAs are polyadenylated except histone mRNAs…
they have unique secondary structure in their 3’ UTRs

Question 9

hnRNPs

Answer 9

heterogeneous nuclear ribonucleoprotein particles)

contribute to polyadenylation, export to cytoplasm, splicing

containing one or more RNA binding domains and often also one or more intrinsically disordered protein domains.

Question 10

Three functions of hnRNPs

Answer 10

• Association with hnRNPs prevent the formation of sequence-specific secondary structures through
  base pairing. The hnRNPs impose a uniform structure that processing enzymes can recognize.
• hnRNPs can regulate pre-mRNA splicing. Many premRNAs can be spliced in more than one way, and
  hnRNPs bound in or near splice sites can promote or repress their use.
• hnRNPs function in RNA transport as some can cycle in and out of the nucleus.

Question 11

Splicing

Answer 11

The GU AG rule…
The A of the Branch point is also highly conserved…

lariat structure
two transesterification reactions

Question 12

small nuclear RNAs (snRNAs)

Answer 12

U1 snRNA: bind with 5’splicing site (GU)
U2 snRNA: bind with branch point

Question 13

Spliceosome

Answer 13

U1U2
U4/5/6
U1/4 leave
U2/5/6: 2 transesterification

no ATP input

Question 14

exception for splicing

Answer 14

self-splicing: most plant
Au….AC introns (not GU…. AG)
trans-splicing

Question 15

rRNA process

Answer 15

snoRNAs

Question 16

tRNA process

Answer 16

remove 5’end
short segment at loop removed
CCA add to 3’end
extensive modification

Question 17

exon definition

Answer 17

SR proteins: bind with ESE
interact with U2AF65 AF35 U1
U2AF65 AF35 interacts with U2
AF35 bind the splice site

Question 18

sxl-lethal

Answer 18

female sxl protein bind to pre-mRNA before exon3, female exclude exon3
exon 3 have inframe stop codon: not functional

Question 19

sxl-lethal with Tra

Answer 19

female sxl protein bind to Tra pre-mRNA before exon 2, female exclude exon2
exon 2 have inframe stop codon: not functional
only female produce Tra protein, activate the splicing site at the end of exon 4 of dsx
female: dsx 3/4 with poly A
male: dsx 3/5

Question 20

affext exon definition

Answer 20

affect splicing site
produce new site
interfere with SR protein, cause exon skipping

Question 21

RNA editing

Answer 21

A to I
C to U

Question 22

protein inport

Answer 22

cargo protein has NLS
bind with importin in cytoplasm
go into nucleus
active RAN-GTP replace importin
back to cytoplasm
RNA-GDP

Question 23

protein export with exportin 1

Answer 23

cargo protein has NES
exportin bind with RAN-GTP go into nucleus
bind cargo
go out to cytoplasm
RNA-GDP

Question 24

RNAs require RAN

Answer 24

tRNA ribosomal subunit specific mRNA with hnRNP

Question 25

RAN-independent

Answer 25

NXF1/NXT1 bind with SR protein go out nucleus

Question 26

RNA remodel

Answer 26

in nucleus: CBC PABPN1 in cytoplasm eIF4E PABPC1

Question 27

SR protein mediate RNA export

Answer 27

Npl3 (SR protein) Glc7 (phosphorase, facilitate binding of the transporter) NXF1/NXT1 Sky 1 Kinase: phosphorylates Npl3

Question 28

RNA survellience

Answer 28

Non-sense mediated decay (NMD) stop codon before last exon Non-stop decay lack of stop codon No-go decy stalled because damage or secondary structure

Question 29

Non-sense mediated decay (NMD)

Answer 29

UPF3: part of exon-juction complex go to cytoplasm with mRNA ribosome displace UPF3 but if stop codon downstream UPF3, ribosome fall off mRNA with UPF3 trigger RNA degradation

Question 30

wooble base

Answer 30

G: C U U: A G I: C A U

Question 31

translation iniation

Answer 31

tRNAi Met + eIF2-GTP tRNAi Met + eIF2-GTP +40S+eIF1/3/5/1A (43S) eIF4E: cap-binding protein eIF4G: bind to eIF3, poly A binding protein eIF4A: remove secondary structure eIF4B: enhance A activity mRNA scanning find and bind with AUG:form 48S eIF complex leave (only 1A and 5B-GTP stay) large subunit bind 5B-GDP form 80S

Question 32

translation elongation

Answer 32

EF1alpha -GTP GTP hydrolysis amino acid chain remove to A site EF2-GTP：move to E site

Question 33

translation termination

Answer 33

eRF1 with eRF3-GTP cleavage and release

Question 34

RNA degradation

Answer 34

deadenylation- dependent deadenylation- independent endonuclease- mediate

Question 35

deadenylation- dependent

Answer 35

deadenylase complex DCP1/2 XPN1： 5-3 exosome: 3-5

Question 36

deadenylation- independent

Answer 36

AU rich 3‘UTR Rsp28B and Edc 3 DCP 1/2 XRN 1

Question 37

endonuclease- mediate

Answer 37

endonuclease cut DCP1/2 XRN1 exosome

Question 38

TfR regulation

Answer 38

high iron：degradation low iron：IRE-BP bind with 3‘ UTR IRE block degradation

Question 39

miRNA

Answer 39

Pol II synthesize pri-miRNA dsRNA hairpin Drosha：pre-miRNA exportin 5 take to cytoplasm Dicer cleave one strand bind Argonacute protein in RISC

Question 40

early embryo regulation

Answer 40

CPE cytoplasmic polyadenylation element CPEB bind CPE recruit Maskin Maskin bind with eIF4E prevent eIF4E bind eIF4G prevent recruit ribosome

Question 41

later embryo

Answer 41

CPEB phosphorated release Maskin recruit eIF4 CPSF and PAP lengthen poly A bind with eIF4E eIF4E bind eIF4G recruit ribosome

Question 42

Ferritin

Answer 42

aconitase： activate at low iron bind 5‘UTR IRE block eIF4A and scanning