is mRNA fully produced before it undergoes processing
no - pre-mRNA processing is co-transcriptional so it occurs as the mRNA is being processed
what enables co-transcriptional processing
the C terminals in RNA Pol II host a number of enzymes necessary for processing mRNA
what are the processing steps that pre-mRNA undergoes
1) 5’ capping
2) splicing
3) cleavage
4) polyadenylation
what happens during 5’ capping
- 7-methylguanylate cap added to the 5’ end of RNA to protect it from degradation
- the cap masks the 5’ triphosphate and protects it against exonuclease attack
what does 5’ capping initiate
indicates that the mRNA is ready for translation
what happens during cleavage
1) RNA Pol II transcribes past primary transcript
2) it transcribes a PolyA signal: AAUAAA
3) it then transcribes a GU or U-rich PolyA signal
4) cleavage factors bind both PolyA signals and make a kink in the RNA
5) the RNA is cut between these two regions at the polyA site (so AAUAAA signal is retained, GU is cleaved off)
6) now the mRNA is released from RNA Pol II
what happens during polyadenylation
1) following cleavage, PolyA polymerase bind the cleavage factors that were on the mRNA
2) the polymerase adds a 100-250 nucleotide sequence of adenosines
3) this creates a polyA tail on the mRNA
what is the purpose of the polyA tail
- prevents degradation
- enhances translation
- mRNA nuclear export
what happens during transcription termination
1) RNA Pol II continues transcription even after cleavage of mRNA, so a signal is needed to stop it
2) it recognises a termination site downstream and finally falls off
3) this leaves us with an RNA sequence between the PolyA site and the termination site, which gets degraded
how do we find what mRNA a cell is making
we sequence the mRNA - focusing especially on the polyA tail using a complement polyT probe
what’s a way to use the polyA tail for mRNA sequencing
- nanopore long-read RNA-sequencing: reading a single RNA molecule directly as it passes through a tiny pore, by measuring changes in electrical current
describe how nanopore long-read RNA-sequencing occurs
1) a polyT adapter binds to the RNA and a motor protein attaches
2) RNA is fed into the nanopore
3) as the RNA passes through the pore, an electrical current is detected
4) different RNA sequences block the hole in slightly different ways resulting in different electrical currents to be recorded
5) the signals are processed by software and converted patterns into their respective A, U, G, C sequences to find the mRNA
what is splicing
- the cutting and joining together of RNA
- simple chemical reactions to join exons together and remove introns (usually)
what happens to introns
(usually) they are cleaved off and the exons are glued together to form the final mRNA and protein
what do exons normally do for a protein
one exon usually corresponds to one unique domain of a protein
what conducts splicing
a spliceosome
what forms the spliceosome
- snRNPs: made of protein and snRNA
- other proteins
describe the process of splicing
1) U1 snRNP binds the 5′ splice site by recognising the conserved GU at the start of the intron
2) U2 snRNP binds conserved A at the branch point
3) a U4, U5 and U6 complex binds conserved AG at the 3’ splice site near the branch point
4) the intron is then cut out
5) the exons are joined together
what is special about snRNA
they are RNAs acting as enzymes
describe an example of issue with splicing
missplicing in motor neuron disease
describe missplicing in motor neuron disease
1) a family with motor neuron disease have a heterozygous change in their DCTN1 gene
2) GU in start of intron at end of exon 2 is changed to CU
3) so the U1 snRNP uses a different, upstream GU
4) this results in a larger area spliced off, losing a crucial binding domain in the mRNA
what is alternative splicing
in some cells, the mRNA skip exons or include introns resulting in diversity in proteins produced
what are 50% of genetic diseases linked to
errors in splicing!
is alternative splicing common
yes - 90-95% of our genes undergo alternative splicing
