RNA Metabolism: RNA Processing

Question 1

Processing of eukaryotic Pre-mRNA

Answer 1

1) 5’ end (capped) 2) internal (splicing introns out) 3) 3’end (polyadenylation)

Question 2

What is capping?

Answer 2

capping occurs when ser 5 is phosphorylated; nucleotide is added in wrong direction ( connected via 5 prime end with 3’OH on end); 7 methyl guanine (methylation allows for binding to protective proteins); tethered to ctd tail

Question 3

What is the purpose of capping?

Answer 3

1) protects from exonuclease activity (exonuclease can’t work because of incorrectly placed nucleotide) 2) RNA transport into cytoplasm 3) Translation

Question 4

3’ Polyadenylation

Answer 4

1) cleavage of RNA 10-30n downstream of 3’ UTR 2) addition of A’s (80-250) to generate a poly A tail

Question 5

what are the only mRNAs that have poly A tails?

Answer 5

only those who are produced by RNAPII

Question 6

CPSF

Answer 6

Cleavage/polyadenylation specificity factor (3 subunit protein) recognizes AAUAAA; recruits CtsF (cleavage factors); Recruits PAP (poly A polymerase).

Question 7

AAUAAA

Answer 7

sequence recognized in RNA 3’ UTR that CPSF binds to.

Question 8

What are the two different models of termination in eukaryotes?

Answer 8

Torpedo and allosteric termination.

Question 9

Torpedo Termination

Answer 9

rat1/hxrn2 (exonuclease) tracks along with RNAP,. Pulls RNA out of RNAP. Once it pulls RNA out, RNAP disassociates.

Question 10

Allosteric Termination

Answer 10

Proteins alter conformation of RNAP causing termination

Question 11

Why is it more advantageous to have more introns?

Answer 11

1) Diversity 2) regulation via splicing produces different types of mRNAs and subsequently different proteins 3)

Question 12

Group I/II splicing

Answer 12

Self-splicing; do not require specific factors or ATP; requires two transesterification reactions

Question 13

Group I

Answer 13

self-splicer; uses guanine nucleoside cofactor; occurs in rRNAs of tetrahymena and mitochondrial regions; 1st cleavage performed by external cofactor guanine

Question 14

Group II

Answer 14

self-splicer; uses an A residue within intron= forming a branched lariat structure; found in mitochondria and chloroplast pre-mRNAs; 1st cleavage is carried out by 2’OH of A within intron.

Question 15

Group III

Answer 15

Eukaryotic; spliceosomal introns; the largest class;

Question 16

Group IV

Answer 16

requires for some tRNAs- requires ATP and an endonuclease.

Question 17

Group III mRNA mechanism

Answer 17

Step I: cleavage at the 5’ splice site (GUintron; AGexon) and joining of the 5’ end of the introns to the branch point A within the intron, producing a lariat-like intermediate 2) Cleavage at the 3’ (AGintron; G exon) splice site and simultaneous ligation of the exons, resulting in excision of the intron as a lariat- like structure

Question 18

SnRNP

Answer 18

small nuclear ribonucleoprotein particles; involved in splicing;

Question 19

What makes up a spliceosome?

Answer 19

SnRNPs and pre-mRNA

Question 20

Model of snRNP mediated splicing of pre-mRNA

Answer 20

1) U1 finds 5’ GU exon-intron junction (recruited by ASF/SF2 a SR- Ser-Arg-rich region) 2) U2AF sits on pyrimidine track sits between A and 3’ GU of intron 3) U2AF recruits BBP 4) BBP replaced by U2 loop outed 5) U4 (masks U6’s ability to react with U2)/6 (catalytic activity binds to U2)/5; 5) tripothide interacts with U2 and U1. U1 exits, U4 exits, U6 can now interact with U2. 7) Adenine attacks junction forming lariat

Question 21

How does the spliceosome find the splice site reliably?

Answer 21

1) Coupling with RNAP. Spliceosomal machinery is present at high concentration at poly A signals 2) Associating splicing factors with CTD

Question 22

Exonic splicing enhancers

Answer 22

ESEs; binding sites for SR proteins; SR proteins interact with each other and recruit the splicing machinery.

Question 23

U5

Answer 23

helps in finding other splice sites; helps keep U4 and U6 together.

Question 24

U1

Answer 24

U1SnRNP binds to the 5’ splice junction

Question 25

U2

Answer 25

U2snRNP binds to the intron branch point and facilitates 2’, 5’-phosphodiester bond formation

Question 26

U4

Answer 26

masks the catalytic activity of U6 in the U4/U6/U5 tri snRNP complex;

Question 27

E complex

Answer 27

Early presplicing complex; defines the intron prior to splicing;

Question 28

U6

Answer 28

base pairs with 5’ site (same as U1), after U1 leaves.