lecture 20- RNA processing part 3 Flashcards
snapshot of human genome
~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
the diversity of proteome is not mainly b/c of alternative splicing but b/c of
posttranslational modifications
name 5 reasons for production of alternative mature mRNA’s that increase protein variability
1- trans-splicing (rare)
2- alternative promoter selection
3- alternative tail site selection
4- alternative (cis) splicing
5- RNA editing
describe trans-splicing in nematodes
- 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
describe mechanism of trans-splicing in nematodes
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
describe alternative promoter selection
- ~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
describe alternative tail site selection
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
describe alternative cis splicing
- 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)
in alternative cis splicing, what plays a major role in determining the product?
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
complex transcripts- combination of methods
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
spliceosome and splice site selection
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
alternative splicing is regulated by ___
activators & repressors
how do activators and repressors regulate alternative splicing
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
majority of known activators are ___
SR proteins composed of 2 functional domains:
1- RNA binding domain
2- RS domain (recruitment and positioning of splicing apparatus)
most of the exonic splicing silencers (ESS) and intronic splicing silencers (ISS) are recognized by…
members of herterogenous nuclear rebonucleoprotein (hnRNP) family
alternative splicing is finely regulated by…
trans-acting hnRNP’s and SR protein families
list three mechanisms of RNA editing
1- ADAR
2- CDAR
3- guide RNA-directed insertions and/or deletions
describe ADAR
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)
describe CDAR
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
describe guide RNA-directed insertions and/or deletions1
- 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
describe editosome: 20S protein complex
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
