exam 4- addition of the 5' cap and poly (A) tail, RNA splicing, alternative splicing

primary transcripts are processed into translation-ready mRNAs that contain what 4 things?

- 5' and 3' untranslated regions (UTR)
- an open reading frame in between the UTRs
- 5' methyl cap
- 3' poly(A) tail

YSPTSPS repeat is an unstructured segment in what domain?

c-terminal domain (CTD)

what is phosphorylation of RNA pol II CTD necessary for?

- promoter escape
- as docking sites for RNA processing enzymes (include the following below)
- capping enzyme
- cap binding complex
- cleavage/termination complexes
- RNA splicing factors

the 5' cap

- 7-methylguanosine on 5' end of mRNA
- has a 5',5'-triphosphate linkage
- added early in transcription by the cap-synthesizing complex bound to pol II CTD

what does the 5' end do once a cap is added?

- cap-synthesizing complex releases mRNA 5' end
- then binds cap-binding complex on the pol II CTD that assists with splicing and nuclear export of RNA

cap snatching

- viral ribonucleoproteins cleave mature cellular mRNAs downstream of the 5' cap structure
- uses snatched 5' cap RNA as a primer for the nascent viral mRNA

poly(A) tail

- a string of A residues added to the 3' end
- serves as a binding site for specific proteins
- helps protect mRNA from enzymatic destruction
- bacteria mRNAs have this to promote mRNA decay

what cleaves at the poly(A) addition site?

endonuclease

polyadenylate (polyA) polymerase

- catalyzes the addition of A resides to the free 3'-OH group by the reaction
- does NOT require a template

what is RNA splicing?

removes intron sequences and links exon sequences

pre-mRNA introns

- removed by large ribonucleoprotein called spliceosome
- RNA of ribonucleoprotein catalyzes reaction

GU/AG rule

- all sequence in between and including the GU at the intron 5' end and the AG at the intron 3' end is excised
- a splice junction linking the 5' to 3' exon is formed

where is the 5' splice site to GU?

immediately upstream

where is the 3' splice to AG?

imm3diately downstream

what is within the intron?

branch point A

spliceosome

nuclear complex remove intron sequences and ligates exon sequences together 5' to 3'

small nuclear ribonuclear protein (snRNP) complex

more than 100 proteins and RNA function in splicing

what are snRNPs named after?

the 100-200 bp small nuclear RNA (snRNA) within the snRNP

U1 snRNP

- binds to the 5' splice site in RNA
- recruited to 5' splice site in the spliceosome reaction

U2 snRNP

- binds the branch site and aligns it for the 1st splicing reaction
- recruited to branch site A in spliceosome reaction

U4 snRNP

- binds to and sequesters U6 snRNP
- binds to spliceosome reaction complex

U5 snRNP

- aligns the pre-RNA for the 2nd splicing reaction
- binds to spliceosome reaction complex

U6 snRNP

- promotes catalysis of RNA splicing reactions
- binds to spliceosome reaction complex

true or false: spliceosome assembly requires ATP

true

activation of spliceosome

- both U1 snRNP and U4 snRNP exit
- U2, U5, U6 snRNAs base pair to each other

group II and pre-mRNA splicing reaction steps

- mechanism uses 2'-5' phosphodiester bond
1. 2'-OH of branch point A attacks 5'-P (5' splice site) of intron
2. 3'-OH of upstream exon attacks 5'-P of downstream exon (3' splice site"

hemophilia

a splicing defect resulting from an error in selection of a 3' splice site

royal hemophiliacs

99.98% chance the wrong 3' splice site is used

frameshift mutation

- changes how the ribosome reads the open reading frame
- leads to inability to clot blood

alternative splicing

process in which a particular exon may or may not be incorporated into the final mRNA

complex transcripts can have more than one site where poly(A) tails can form...?

poly(A) sites

what PROMOTES splicing by binding exonic splicing enhancers (ESE)?

SR proteins (serine/arginine rich)

what INHIBITS splicing by binding to exonic splicing silencers (ESS)?

heterogenous nuclear ribonuclearproteins (hnRNP)

describe the nuclear export of mRNA

- cap binding complex binds to nuclear pore complex
- mRNA threaded through nuclear pore 5'-3'
- cytoplasmic proteins replace nuclear proteins on mRNA