LEC25: mRNA Processing II Flashcards
what is the 5’ CAP made of?
what bond is formed by its addition?
what is the purpose?
5’CAP is a 7-methyl-guanisine
forms a 5’-5’ phosphate diester bond
makes mRNA look diff from other RNAs, indicates it should be translated
what is the structure of an intron?
begins w/ GU
has an A residue, a branch point, where 2’ to 5’ phosphodiester bond forms when intron is spliced out
concentration of pyrimidines toward downstream exon
has 2 loose consensus sequences
ends w/ AG
what happens if splicing is incorrect by 1 ntd?
get frameshift mutation downstream
what are the consensus sequences required for splicing?
5’ splice site consensus, GT
branch point consensus, A
3’ splice site consensus, AG
size of large intro in muscular dystrophy?
250,000 ntds
how is it possible that huge introns don’t get included in mRNA?
exon definition!
spliceosome recognizes edges of exons, which are much shorter than introns
exon definition allows for protein-protein and protein-RNA interactions
what is an ESE?
exonic splice enhancer sequence
RNA box bound by a class of proteins called SR proteins (rich in Ser and Arg)
the correct 5’ GU and 3’ AG dinucleotides of splice junctions can be reconigzed specifically by splicing machinery on basis of their proximity to exons in which SR proteins are bound
what would happen if there’s a silent mutation in an ESE?
SR protein needs certian consensus sequence in ESE to know where to bind
if change occurs, SR sequence cannot recognize it needs to be
so get problems
where does the first exon start?
w/ the +1 nucleotide, includes the start codon
upstream of start codon is the 5’ UTR
why must splicing be precise?
in order to not disrupt the coding sequence reading frame, which spreads over multiple exons
what does the final exon contain?
the polyadenylation signal
how is it that just 25,000 genes in the human genome, but we have such great complexity?
each gene transcript can undergo alternative splicing, gives multiple mRNA products
what % of transcripts udnergo alternative splicing?
transcripts from >90% of genes undergo alternative splicing
what is the transcriptome? why so big?
all the different number of transcripts that can be produced from a particular genome
per alternative splicing, for humans
what are the functions of SR proteins?
1) **constituitive: **bind EXEs
2) regulated: not always active!
is splicing the same for all parts of the body’s genes?
no; there is tissue specificity to alternative splicing
what is the most common form of alternative splicing?
other forms?
most common: exon skipping
others: alternative 5’ splice site, alternative 3’ splice site, intron inclusion
what is an ESS?
exonic splice silencers
RNA boxes in exons that bind SR proteins to suppress splicing of an exon, such that it’s not included in final mRNA product
what are ISE, ISS?
deep intronic mutations - intronic splice enhancers and intronic splice silencers - that tell gene to splice out or keep an intron
what is the big controller of splicing?
SR proteins!
how much of genetic disease is caused by splicing issues?
15%!
what about silent mutation in coding sequence might be involved w/ splicing?
change in the 3rd ntd position of a codon which doesn’t change encoded amino acid now thought to be involved in splicing regulation
what are the reactions of splicing?
2 transesterification reactions:
1) attack of the branch point A to OH group on 5’ splice site, makes 2’ to 5’ phosphodiester bond
2) attack of the 3’ OH of upstream exon on the 3’ splice site; joins the upstream and downstream exons in a 3’ to 5’ phosphodiester bond
what is lariat RNA
the form of the intron sequence that is spliced out and degraded by ribonucleases
what’s the typical poly-a signal?
AAUAAA
what is an endonuclease?
ribonuclease that cuts RNA internally
cuts 20-30 ntds downstream of the Poly-A signal
results in a free 3’OH
how does the poly-A rxn occur?
1) **highly conserved **poly-A signal (AAUAAA) at end of last exon
2) endonuclease cuts 20-30 ntds downstream of poly-A signal
results in free 3’OH
3) poly-A polymerase adds **poly-AAAAA to 3’ OH **just generated
what protects the poly-A tail when the mRNA is exported to the cytoplasm?
**poly-A binding proteins **coat the poly-A tail
protects mRNA from exonucleases
what is the EJC?
what is its relation to nonsense mediated decay?
exon junction complex
deposit of proteins where exons are knit together
if there’s a **premature stop codon **b/c of nonsense mediated decay, and there’s an **EJC **downstream of the too-early stop codon,
**when ribosome encounters stop codon, EJC transmits a signal, mutated RNA gets degraded **
what happens to mRNAs in cytoplasm? what does it influence?
mRNAs are translated & eventually degraded in cytoplasm
rate of degradation is a factor that determines the amount of translation from an mRNA
decay of mRNA allows recycling of ntds, enables changes in set of genes being expressed in response to changing conditions
what does nonsense-mediated decay pathway do?
rapid, targeted degradation of mRNAs containing a nonsense codon
ensures that defective mRNAs w/ a premature stop codon are dealt w/
