lecture 20- RNA processing part 3

Question 1

snapshot of human genome

Answer 1

~100,000 human genes were expected
- found to be much lower, 18-20,000 of genes are protein-coding genes (1.5% of human genome) - however, the number of diff proteins greatly exceeds the number of identified genes…b/c of splicing, alternative splicing produce many diff mRNA’s which produce many diff proteins
- half of human genome composed of transposons

Question 2

the diversity of proteome is not mainly b/c of alternative splicing but b/c of

Answer 2

posttranslational modifications

Question 3

name 5 reasons for production of alternative mature mRNA’s that increase protein variability

Answer 3

1- trans-splicing (rare)
2- alternative promoter selection
3- alternative tail site selection
4- alternative (cis) splicing
5- RNA editing

Question 4

describe trans-splicing in nematodes

Answer 4

• trans b/c involves 2 diff mRNA’s
• not known to occur in humans or most other eukaryotes
• predominant mechanism of mRNA maturation in nematode worms
• in trans-splicing, there is no lariat, but a Y-shaped structure
• trans-splicing is catalyzed by spliceosomes, including U2, U4, U5, and U6 snRNPs, but not U1
• exons from diff mRNA’s are fused together, producing alternative mRNA’s

Question 5

describe mechanism of trans-splicing in nematodes

Answer 5

in nematodes, a short 5’ capped leader sequence (SL RNA) is spliced onto a primary transcript (pre-mRNA) to produce mature mRNA

• branch point -OH on the pre-mRNA attacks the 5’ splice site on the SL RNA
• the free hydroxyl at the 5’ splice site attacks the 3’ splice site, forming a capped mRNA

Question 6

describe alternative promoter selection

Answer 6

• ~18% or more of all human genes (many genes in Alzheimers use alternative promoter selection)
• RNA polymerase binding to certain promoter due to regulatory proteins
• impt mechanism for transcriptome diversity and the regulation of gene expression
• many promoters in a gene, diff exons spliced b/c of it

Question 7

describe alternative tail site selection

Answer 7

Alternative Polyadenylation (APA)

• 70% of human genes show alternative poly A site, and 50% have 3 or more polyadenylation sites
• ex: 2 diff transcripts produced from same gene b/c of polyadenylation site (distinct mRNA isoforms with different 3’ untranslated regions

Question 8

describe alternative cis splicing

Answer 8

• produce the most variety of mRNA of any of the methods we have talked about
• increases complexity of genome and provides opportunities for regulation at the level of pre-mRNA processing
• at least 90% of human genes undergo alternative cis splicing
• diff sequences produced from same gene –> leading to diff proteins (mechanisms not well understood)
• in extreme cases, genes could produce thousands of alternative products (ex: DSCAM from Drosphilia…over 38,000 isoforms of DSCAM protein)

Question 9

in alternative cis splicing, what plays a major role in determining the product?

Answer 9

nucleotide sequences within the intron and at the borders between introns and exons play an impt role in determining whether an exon is included in the mature mRNA

Question 10

complex transcripts- combination of methods

Answer 10

complex transcripts = more than one site for cleavage and polyadenylation, or for alternative splicing patterns, or both
- alternative processing of the rat calcitonin gene transcript produces 2 diff calcitonin protein sequences encoded by the same gene

Question 11

spliceosome and splice site selection

Answer 11

in animal pre-mRNA’s: exons about 150 nucleotides and introns over 3000 (50-20,000 nt)
- correct sites chosen b/c of:
1- co-transcriptional “loading” of splicing machinery
2- binding of SR (serine-arginine rich) proteins to exonic splicing enhancers (ESEs) - ESE’s help to recruit and position proteins at correct site

Question 12

alternative splicing is regulated by ___

Answer 12

activators & repressors

Question 13

how do activators and repressors regulate alternative splicing

Answer 13

activators (trans-acting) bind to exonic or intronic splicing enhancers (cis-acting ISE or ESE)
- repressors bind to exonic or intronic splicing silencers (ESS or ISS)
- if more repressors –> not spliced
- if not activator —> not spliced
- activators/repressors determine which part will be spliced

Question 14

majority of known activators are ___

Answer 14

SR proteins composed of 2 functional domains:
1- RNA binding domain
2- RS domain (recruitment and positioning of splicing apparatus)

Question 15

most of the exonic splicing silencers (ESS) and intronic splicing silencers (ISS) are recognized by…

Answer 15

members of herterogenous nuclear rebonucleoprotein (hnRNP) family

Question 16

alternative splicing is finely regulated by…

Answer 16

trans-acting hnRNP’s and SR protein families

Question 17

list three mechanisms of RNA editing

Answer 17

1- ADAR
2- CDAR
3- guide RNA-directed insertions and/or deletions

Question 18

describe ADAR

Answer 18

RNA editing by base deamination- common in mammals and their viruses
- Adenosine Deaminase Acting on RNA

• coverts adenosine (A) –> inosine (I)
• glutamine to arginine in protein channel, much more difficult for protein transport - the arg residue results in a protein that is much more efficient for Ca2+ transport (human glutamate receptor calcium channel)

Question 19

describe CDAR

Answer 19

RNA editing by base deamination- common in mammals and their viruses
- Cytidine Deaminase Acting on RNA

• converts cytosine (C) to uracil (U)
• CAA –> UAA (stop codon- creates truncated form of protein)
• ApoB100 impt for transport of cholesterol

Question 20

describe guide RNA-directed insertions and/or deletions1

Answer 20

• mechanism used in mitochondria of trypanosomes
• guide RNA (gRNA) as a template to add or remove bases from mRNA
• the pre-mRNA’s are edited after synthesis by an enzyme that inserts some U residues and deletes others

Question 21

describe editosome: 20S protein complex

Answer 21

endonucleases cut at the mismatch point between the gRNA and the unedited transcript
- TUTase (terminal uridyltransferase): adds U residues from UTP to the 3’ end of mRNA
- U-specific exoribonuclease: removes any unpaired U residues
- RNA ligase