VL 23 (Michael Lennhard)

Question 1

Important definitions

Answer 1

pre-mRNA
The nuclear transcript that is processed
by modification and splicing to give an mRNA.
RNA splicing
The process of excising introns
from RNA and connecting the exons into a continuous mRNA

Picture:
1. transcription by RNA Pol II
2. add cap at 5 ́ end + poly(A) at 3 ́ end
3. splicing (remove introns + joining up of exons)
4. transport of mature mRNA through nuclear pore complex (NPC) → cytoplasm
5. ribosome binding → translation

Question 2

The 5 ́-end of eukaryotic mRNA is capped:

Answer 2

• 5 ́ cap formed by G-adding to terminal transcript base via 5 ́-5 ́ link
• capping during transcription; important for transcription pausing release
• cap structure recognized by protein factors to influence mRNA stability, splicing, export, translation

capping:
* 3 phosphate groups between two 5 ́ ribose ends
* release of RNA Pol II + transition into fully productive, elongating mode
* capping is stimulated by some PTMs, e.g. Serine-5-phosphorylation on C-terminal domain of RNA Pol II
* cap structure is recognized by cap-binding proteins → influence mRNA stability etc.
* constitutive process
* eukaryotic, premRNAs
* no regulation

Question 3

Nuclear splice sites are short sequences

Answer 3

GU-AG rule (GT-AG rule in terms of DNA sequence):
describes the requirement for these constant dinucleotides at first, last two intron positions in pre-mRNAs

Question 4

Pre-mRNA Splicing Proceeds Through a Lariat

Answer 4

• conserved sequence in middle of intron: A = branch site
• upstream of splice site = polypyrimidine tract (CT-rich)
• additional ends of nuclear introns; less common
• process of 5 ́-2 ́ bond formation: trans-esterification
• joined up exons after intron is released as lariat → debranched lariat → degraded
• splicing is temporally and functionally coupled with various processes in gene expression
• first introns spliced while transcription occurs
• part of this integration is due to the C-terminal domain on RNA-Pol II
• EJC
  –> signals successful splicing
  –> in important quality control mechanism for mRNA

Question 5

Splicing is temporally and functionally coupled with multiple steps in gene expression

Answer 5

• splicing
  –> during/after transcription
  –> connected to mRNA export, stability control o can influence mRNA translation in cytoplasm
• transcription, splicing machineries are physically, functionally integrated
• exon junction complex (EJC):
  protein complex that assembles at exon-exon junctions during splicing and assists in RNA transport, localization,
  degradation
• nonsense-mediated mRNA decay (NMD): pathway that degrades mRNA with nonsense mutation prior to last exon

Picture
* mRNA quality control step (see EJC) linked to nonsense-mediated mRNA decay (NMD)
* process
–> fully processed mRNA after splicing
–> transcription error/DNA mutation
→ premature stop codon o premature stop codon in penultimate exon → exon-exon junction (marked by EJC) behind premature stop codon
–> normally - first translation round (maybe nucleus): ribosome
displaces EJC
–> premature stop codon → ribosome disassembles at/after stop codon → EJC not displaced + EJC recruits factor Upf o Upf recruits decapping enzymes (DCP) → decap mRNA → degradation
* premature stop codons lead to truncated protein versions → act in a dominant negative manner (poison / inhibit activity of normal protein versions)
➔ quality control mechanism evolved to get rid of faulty mRNAs with premature stop codons to prevent truncated protein versions from accumulating

Question 6

Alternative Splicing Is a Rule, Rather
Than an Exception, in Multicellular Eukaryotes

Answer 6

Left
* blue: constitutive exons; always included in mRNA
* pink: alternative exons
* alternative 5/3 ́ splice sites → longer/shorter exons
* skipped alternative exons

right:
* used to control gene expression
* sex determination in D. melanogaster
* tra: transformer gene → transcribed into pre-mRNA → spliced without SxI + premature stop codon
or with SxI but without premature stop codon
* presence/absence of SxI
–> made in females→full-length protein version
–> males → truncated protein version
* tra = splicing regulator; + tra2 act on double sex (Dsx) pre-mRNA → different spliced versions which interact with other genes

Question 7

The 3 ́ ends of mRNAs are generated by cleavage and polyadenylation:

Answer 7

• sequence AAUAAA = cleavage signal → generate 3 ́-mRNA end →polyadenylated
• reaction requires protein complex that contains a specificity factor = endonuclease + poly(A) polymerase
• specificity factor + endonuclease cleave RNA downstream of AAUAAA
• specificity factor + poly(A) polymerase add 2ßß A residues processively to 3 ́ end
• poly(A) tail controls mRNA stability + influences translation
• cytoplasmic polyadenylation play major role in Xenopus embryonic development
• polyadenylation linked to transcription termination
• no dedicated terminator sequences (eukaryotes)
• sequence AAUAAA trigger cleavage of nascent pre-mRNA
• endonuclease cleaves nascent pre-mRNA
• sequence AAUAAA recruits poly(A) polymerase (adds poly(A) tail)
• cleavage → free, non-modified non-capped 5 ́ end → targeted by
  exonucleases → chase after transcribing RNA Pol, contributes to transcription termination
• AAUAAA recruits certain factors
  –> cleavage polyadenylation specificity factor (CPSF) thought to
  includes endonuclease + recruit poly(A) polymerase
  –> cleavage stimulating factor (CstF)
  –> 1st step: 3 ́-OH group after cleavage → mechanism (see picture starts)
  –> PBPs help to maintain poly(A) polymerase in a processive
  state → complex dissociate → no further A-addition happens