LEC25: mRNA Processing II

Question 1

what is the 5’ CAP made of?

what bond is formed by its addition?

what is the purpose?

Answer 1

5’CAP is a 7-methyl-guanisine

forms a 5’-5’ phosphate diester bond

makes mRNA look diff from other RNAs, indicates it should be translated

Question 2

what is the structure of an intron?

Answer 2

begins w/ GU

has an A residue, a branch point, where 2’ to 5’ phosphodiester bond forms when intron is spliced out

concentration of pyrimidines toward downstream exon

has 2 loose consensus sequences

ends w/ AG

Question 3

what happens if splicing is incorrect by 1 ntd?

Answer 3

get frameshift mutation downstream

Question 4

what are the consensus sequences required for splicing?

Answer 4

5’ splice site consensus, GT

branch point consensus, A

3’ splice site consensus, AG

Question 5

size of large intro in muscular dystrophy?

Answer 5

250,000 ntds

Question 6

how is it possible that huge introns don’t get included in mRNA?

Answer 6

exon definition!

spliceosome recognizes edges of exons, which are much shorter than introns

exon definition allows for protein-protein and protein-RNA interactions

Question 7

what is an ESE?

Answer 7

exonic splice enhancer sequence

RNA box bound by a class of proteins called SR proteins (rich in Ser and Arg)

the correct 5’ GU and 3’ AG dinucleotides of splice junctions can be reconigzed specifically by splicing machinery on basis of their proximity to exons in which SR proteins are bound

Question 8

what would happen if there’s a silent mutation in an ESE?

Answer 8

SR protein needs certian consensus sequence in ESE to know where to bind

if change occurs, SR sequence cannot recognize it needs to be

so get problems

Question 9

where does the first exon start?

Answer 9

w/ the +1 nucleotide, includes the start codon

upstream of start codon is the 5’ UTR

Question 10

why must splicing be precise?

Answer 10

in order to not disrupt the coding sequence reading frame, which spreads over multiple exons

Question 11

what does the final exon contain?

Answer 11

the polyadenylation signal

Question 12

how is it that just 25,000 genes in the human genome, but we have such great complexity?

Answer 12

each gene transcript can undergo alternative splicing, gives multiple mRNA products

Question 13

what % of transcripts udnergo alternative splicing?

Answer 13

transcripts from >90% of genes undergo alternative splicing

Question 14

what is the transcriptome? why so big?

Answer 14

all the different number of transcripts that can be produced from a particular genome

per alternative splicing, for humans

Question 15

what are the functions of SR proteins?

Answer 15

1) **constituitive: **bind EXEs
2) regulated: not always active!

Question 16

is splicing the same for all parts of the body’s genes?

Answer 16

no; there is tissue specificity to alternative splicing

Question 17

what is the most common form of alternative splicing?

other forms?

Answer 17

most common: exon skipping

others: alternative 5’ splice site, alternative 3’ splice site, intron inclusion

Question 18

what is an ESS?

Answer 18

exonic splice silencers

RNA boxes in exons that bind SR proteins to suppress splicing of an exon, such that it’s not included in final mRNA product

Question 19

what are ISE, ISS?

Answer 19

deep intronic mutations - intronic splice enhancers and intronic splice silencers - that tell gene to splice out or keep an intron

Question 20

what is the big controller of splicing?

Answer 20

SR proteins!

Question 21

how much of genetic disease is caused by splicing issues?

Answer 21

15%!

Question 22

what about silent mutation in coding sequence might be involved w/ splicing?

Answer 22

change in the 3rd ntd position of a codon which doesn’t change encoded amino acid now thought to be involved in splicing regulation

Question 23

what are the reactions of splicing?

Answer 23

2 transesterification reactions:

1) attack of the branch point A to OH group on 5’ splice site, makes 2’ to 5’ phosphodiester bond
2) attack of the 3’ OH of upstream exon on the 3’ splice site; joins the upstream and downstream exons in a 3’ to 5’ phosphodiester bond

Question 24

what is lariat RNA

Answer 24

the form of the intron sequence that is spliced out and degraded by ribonucleases

Question 25

what’s the typical poly-a signal?

Answer 25

AAUAAA

Question 26

what is an endonuclease?

Answer 26

ribonuclease that cuts RNA internally

cuts 20-30 ntds downstream of the Poly-A signal

results in a free 3’OH

Question 27

how does the poly-A rxn occur?

Answer 27

1) **highly conserved **poly-A signal (AAUAAA) at end of last exon
2) endonuclease cuts 20-30 ntds downstream of poly-A signal

results in free 3’OH

3) poly-A polymerase adds **poly-AAAAA to 3’ OH **just generated

Question 28

what protects the poly-A tail when the mRNA is exported to the cytoplasm?

Answer 28

**poly-A binding proteins **coat the poly-A tail

protects mRNA from exonucleases

Question 29

what is the EJC?

what is its relation to nonsense mediated decay?

Answer 29

exon junction complex

deposit of proteins where exons are knit together

if there’s a **premature stop codon **b/c of nonsense mediated decay, and there’s an **EJC **downstream of the too-early stop codon,

**when ribosome encounters stop codon, EJC transmits a signal, mutated RNA gets degraded **

Question 30

what happens to mRNAs in cytoplasm? what does it influence?

Answer 30

mRNAs are translated & eventually degraded in cytoplasm

rate of degradation is a factor that determines the amount of translation from an mRNA

decay of mRNA allows recycling of ntds, enables changes in set of genes being expressed in response to changing conditions

Question 31

what does nonsense-mediated decay pathway do?

Answer 31

rapid, targeted degradation of mRNAs containing a nonsense codon

ensures that defective mRNAs w/ a premature stop codon are dealt w/