mRNA processing Flashcards
When does mRNA processing occur?
Cotranscriptional, occurs at same time
Order of mRNA processing
Capping
Splicing
Polyadenylation
What is mRNA called directly after transcription?
pre-mRNA
Where does mRNA processing occur?
In the nucleus
What happens to G residue in capping?
Methylated and binds to protein complex called cap binding complex (methyl guanosine cap)
What are the roles of 5’ capping (4)?
Protect 5’ of mRNA from degradation by exonucleases
Improve ribosomal recognition for translation
Provide scope for translation regulation
Recognised by export machinery - helps mRNA move out of nucleus
What is the 5’ cap?
Altered nucleotide on 5’ end
What happens in capping?
GMP nucleotide/ G residue added to 5’ end of pre-mRNA by 5’-5’ triphosphate bond
What is unique about the 5’ end linkage?
5’-5’ bond
What does splicing do to exons and introns?
Excision of introns and fusion of exons
What directs splicing?
Splicesome
What does the spliceosome consist of?
5snRNAs bound to 100 splicing factors - forms complexes known as snRNPs
What sort of reaction occurs in splicing?
snRNA mediated transesterification reaction
Causes 2 sequential breakages and rejoining of RNA sugar phosphate backbone
Where are splice sites?
5’ and 3’ ones at border of each intron/exon
What group is at the end of a 5’ cap , why is this important?
3’OH, not recognised by exonucleases
What do snRNAs do?
Complementary sequence to splice site
Recruits components of spliceosome - RNA protein interactions between spliceosome and preRNA allows introns to be spliced
What is the process of splicing?
2’ OH of adenosine in intron (branch point) nucleophilic attack on 5’ exon/intron border
Transesterification reaction causes lariat intron joined with 3’ exon and release of 5’ exon
3’ OH of 5’ exon attacks lariat intron/3’ exon junction
Transesterification then fuses 5’ and 3’ exons.
What is found at the branch point?
2’OH of an adenosine
What are snRNPs?
Small nuclear RNAs (snRNAs) bound to specific proteins
Ribonucleoproteins snRNPs
What is at 5’ end of intron?
GU
What is at 3’ end of intron?
AG
What happens at exon exon junctions after splicing?
Marked by deposition of exon junction proteins/complex
What is nonsense mediated decay?
Surveillance mechanism in which exon junction proteins act as markers to identify faulty mRNA
How does nonsense mediated decay protected mRNA?
Exon junction complex downstream of stop codon recognised
Indicates premature stop codon so faulty mRNA degraded
After what point should you no longer find exon junction complexes?
After normal stop codon.
What does alternative splicing allow?
Tissue specific gene expression, several proteins to be made from single gene
What correlates with increased complexity of organism?
More introns per gene and thus greater expansion of proteome.
When/where can alternative splicing occur?
In different cell types or response to different signals
How can alternative splicing be made to include certain exon?
Enhancer proteins bind enhancer sequence in exons, make splice site more attractive.
More likely to include the exon in mRNA
How can alternative splicing prevent splicing of a gene?
Proteins bind to silencer region prevent spliceosome associating with splice site (weaken splice site)
What happens when enhancer proteins associated with mRNA?
Splicing factors more likely to recognise 3’ and 5’ splice sites that flank an exon
How can an exon be skipped?
Negative factors bind specific exon sequences prevent recognition of 3’ and 5’ splice sites flanking exon
What are 2 steps of polyadenylation?
premRNA cleaved 20 bases downstream by endonucleases
Poly(A) polymerase adds Poly(A) tail to 3’ end
What does polyadenylation consensus sequence signal (AAUAAA)?
Signals cleavage of premRNA 20 bases downstream
Signals polyadenylation
What are cleave and polyadenylation machinery associated with?
AAUAAA and DSE sequence (second signal after cleavage site)
How does polyA tail form
Poly(A) polymerase adds poly(A) tial to 3’ end
Donated by ATP which is then covered by Poly(A) binding proteins
How many residues is poly(A) tail?
250 adenosine resiudes
What donates adenosine residue for poly (A) tail?
ATP
What happens after poly A tail formed?
Covered by poly(A) binding proteins
What enzyme catalyses poly(A) tail formation?
Poly(A) polymerase
What are the roles of Poly(A) tail (3)?
Uniform 3’ end for nuclear cytoplasmic transport, stability, translatability
Makes mRNA more stable because must be degraded by exonucleases before they reach coding mRNA, so more time for this to be translated
Cap and poly (A) binding proteins interact, forming structure that recruits ribosome to mRNA- enhances translation
How can mutations affect splice sites?
Lead to reduced/abolished splicing of some introns, or faulty mRNAs that contian intronic sequences.
What can mutations affecting poly(A) signals do?
Increase/reduce cleavage and polyadenylation so total ouput of mRNA affected
(i.e. poly(A) machinery may not recognise AAUAAA sequence)
How can mRNA presence in the cell be regulated?
Transcription of siRNAs and miRNAs that target and break down mRNA in the cytosol.
How do siRNAs break down mRNA?
siRNA originates as a long RNA strand that is double stranded.
Dicer enzymes then catalyse the production of siRNAs, enabling the siRNA to associate with proteins to form a RISC complex that is single stranded.
The siRNA then binds to the target mRNA and induces cleavage. So it cannot be translated.
How do miRNAs break down mRNA?
Less specific short RNA sequences that bind to mRNA strands to physically block it from translation.
