Post-Translational Gene Control (L12)

Question 1

3 functions of 5’ cap

Answer 1

1. stabilizes mRNA (bound by cap-binding complex - protection from degradation)
2. translation initiation
3. nuclear export

Question 2

heterogenous nuclear RNAs (hnRNAs)

Answer 2

non-coding nuclear RNAs - mainly pre-mRNAs, but also tRNAs, rRNAs, etc.

Question 3

what do hnRNA’s bind?

Answer 3

hnRNP’s (heterogeneous ribonucleoprotein particles)

Question 4

RNP RRM (RNA recognition motif) structure

Answer 4

extensive B sheets w/ mainly basic (+) aa’s -> interacts w/ negative phosphate backbone of mRNA

Question 5

3 functions of RNP’s

Answer 5

1. prevent formation of secondary structures in RNA
2. RNA splicing
3. transport of mRNA

Question 6

structure of splice sites

Answer 6

5’-GU……..A……pyrimidine-rich region…..AG -3’

A = branch point - this nucleotide mediates splicing process

Question 7

describe splicing reactions

Answer 7

• 2’-OH of branch point A-> nucleophilic attack of phosphodiester bond b/w intron and exon (transesterification)
• creates 3’-OH that participates in 2nd nucleophilic attack on splice site -> creates spliced exons + excised lariat intron

Question 8

what composes functional unit of splicing?

Answer 8

snRNA + snRNP

Question 9

what do U1 and U2 snRNA’s bind?

Answer 9

U1: binds 5’ splice site
U2: binds sequence including branch point

Question 10

what makes it possible for branch point A to participate in transesterification reactions?

Answer 10

branch point not included in complementary binding -> A sticks out -> 2’-OH free to act as a nucleophile

Question 11

what proteins compose the spliceosome?

Answer 11

U1, U2, U4, U5, U6

Question 12

function of phosphorylated CTD

Answer 12

carrier of many proteins used for post-translational processing - snRNPs, 5’ cap, poly A tail proteins

Question 13

what is ESE?

Answer 13

exon splicing enhancer - 6 bp long consensus sequence bound by SR proteins, interact w/ each other to help anchor U1 on 5’ splice site and anchor U2 factors

Question 14

what are SR proteins?

Answer 14

proteins rich in Ser and Arg

Question 15

how are spliceosomes laid down?

Answer 15

cross-exon recognition complex - not put down all in one piece/ at one time

Question 16

two examples of significance of cross-exon recognition complex/ alternative splicing

Answer 16

1. dsx in drosophila (TRA2 lays down part of spliceosome, but needs TRA - TRA only in females)
2. B-thallassemia caused by alt splicing defect

Question 17

another name for intron

Answer 17

IVS - intervening sequence

Question 18

mechanism of polyadenylation

Answer 18

1. 3’ end of pre-mRNA has polyA signal (AAUAAA) - to tell cell to put down a polyA tail
2. many proteins used to recruit CPSF and CSI
3. CPSF and CSI bind consensus sequence
4. facilitate binding of CFII
5. facilitate binding of PAP
6. breaks b/w polyA signals
7. polyA polymerase picks up, starts adding A’s on 3’ end slowly
8. after 12 A’s, PABPII binds polyA tail -> rapid synthesis

Question 19

3 functions of PABPII

Answer 19

1. protects 3’ end of mRNA (prevents nuclease degradation)
2. increases translation efficiency
3. participates in nuclear transport

Question 20

EJC

Answer 20

exon junction complex - proteins on 5’ end of each spliced jxn -> marks all jxns b/w exons

Question 21

structure of processed mRNA

Answer 21

5’ cap… 5’ UTR… cds… 3’ UTR…polyA tail

Question 22

composition of NPCs (nuclear pore complexes)

Answer 22

nucleoporins

Question 23

function of FG nucleoporin central core of NPC

Answer 23

act as a barrier for the diffusion of large macromolecular complexes

Question 24

function of REF (RNA export factor)

Answer 24

on mature mRNA to help nucleoporin recognize something that needs to be transported to cytoplasm

Question 25

what modifications are made to mRNA after it has been transported to cytoplasm?

Answer 25

1. cap-binding complex replaced with eIF4E | 2. PABPII replaced by PABPI

Question 26

what happens to PABPII once replaced by PABPI in cytoplasm?

Answer 26

some recycled back into nucleus to mediate more post-transcriptional processing

Question 27

how do 5' cap and polyA tail facilitate translation?

Answer 27

PABPI on polyA tail binds eIF4G, which binds eIF4E (replaced cap) -> forms loop

Question 28

miRNA vs. siRNA: binding of target?

Answer 28

miRNA: imperfect binding siRNA: perfect binding

Question 29

miRNA vs. siRNA: function?

Answer 29

miRNA: translation inhibition siRNA: RNA cleavage

Question 30

miRNA vs. siRNA: how many mRNAs can one bind?

Answer 30

miRNA: one recognizes many mRNAs siRNA: one recognizes only one mRNA

Question 31

miRNA vs. siRNA: where does it bind?

Answer 31

miRNA: binds 3' UTR siRNA: binds anywhere

Question 32

miRNA vs. siRNA: endogenous or exogenous?

Answer 32

miRNA: endogenous (encoded in genome) siRNA: exogenous (introduced)

Question 33

miRNA (miR) processing

Answer 33

1. after transcribed, pri-miR forms hairpin loop in nucleus bound by drosha and pasha (DGCRB) 2. now pre-miR 3. exported to cytoplasm 4. bound by dicer and TRBP 5. now ds miR

Question 34

function of drosha vs. dicer

Answer 34

``` drosha = nuclear RNAse dicer = cytoplasmic RNAse ```

Question 35

what happens to mature ds miR?

Answer 35

Argonaute protein picks up one strand of miR -> forms RISC -> this binds 3' UTR to bind target mRNA -> inactivates translation and promotes deadenylation

Question 36

where did the miRNA mechanism evolve from?

Answer 36

immune response to foreign invaders in primitive organisms

Question 37

most extreme way to regulate mRNA expression

Answer 37

degradation - can chop from sides or from middle

Question 38

mRNA surveillance

Answer 38

EJC works with RF to say something is wrong -> nonsense mediated decay -> detaches EJC as ribosome slides along mRNA

Question 39

TOR (target of rapamycin) pathway

Answer 39

TOR kinase inhibits 4E-BP which inhibits eIF4E binding to 5' cap

Question 40

what are ferritin and transferrin regulated by?

Answer 40

IRE-BP (iron response element binding protein)