SONENBERG Flashcards
RNA splicing
how was mRNA processing discovered
Discovered in bacteria (E.coli)
look at the mRNA in the eukaryote
found that the mRNA in the nucleus was about 5x longer than the mRNA in the cytoplas
what are some elements present on the mRNA
nuclear mRNA from eukaryotes: cut at 5’ end and has a poly A tail at 3’ end in the nucleus.
The cap and poly A tail are present in both nuclear and cytoplasmic RNA
how was splicing discovered through R loop hybridization
R-loop hybridization (seen in adenovirus under electron microscope)
ssDNA and RNA can form a hybrid (In prokaryotes, there is no looping)
R-loops of ssDNA can form when you hybridize RNA and DNA.
The looping sequence = DNA (cannot hybridize to RNA = not complementary)
dsDNA and RNA also have R-loops of dsDNA.
Electron microscopy of splicing
Nobel prizes
Philip Sharp and Richard Roberts (physiology/medicine 1993): Splicing of mRNA
Tom Cech and Sidney Altman (chemistry 1989): Ribosomal RNA self-splicing
what are the two steps of mRNA splicing
how does rRNA and tRNA undergo splicing
rRNA is self splicing, no need for other proteins
tRNA is enzymatic: endonuclease, kinase, ligase, phosphatase etc
how does group 1 self splicing work
Guanosine (exogenous) attacks exon intron junction → second attack.
End up with a linear structure.
how does group 2 self splicing work
Endogenous A (like mRNA) attacks the junction → becomes part of the lariat.
Second attack = the 3’OH on the junction.
what is spliceosome splicing
The spliceosome must have evolved from self-splicing RNA (also no enzymes and energy needed).
for nuclear mRNA
characteristics of the 7 methyl guanosine cap
Important for translation and splicing.
No CAP = no splicing and no transport.
contains 7-mG: the 7th position of G is methylated
without the methyl it doesn’t work; invert linkage
connected to the mRNA by a 5’ triphosphate linkage (5’-5’)
unique because the nt are typically attached by 3’ phosphodiester bond.
Capping protects against exonucleases (due to methylation)
diagram of pre-mRNA processing
what are the conserved sequences in introns that indicate for splicing?
recognized by splicing factors = how machinery knows where to splice
GU-AG rule (cis-acting marker)
GU at the beginning of the intron (5’ splice site)
AG at the end of the intron (3’ splice site)
A stretch of pyrimidines precedes the AG.
A branch site.
Yeast branch site = TACTAAC (100% conserved)
what features of core splicing signals influence regulation
Strength of splicing signals
Spacing between signals
Accessibility: presence of RNA structure prevents binding of splicing factors
Core signals lack sufficient information required to specify correct pairs of splice sites and to regulate alternative splicing
need additional cis elements.
what are additional cis elements that regulate splice site selection
what do trans acting factors do
regulate splicing via binding enhancers and silencers = family of proteins with similar motifs ↑
combinations of proteins make the specific mRNA splicing
SR = Ser/Arg-repeat protein
hnRNP = heterogeneous nuclear ribonucleoprotein (rich in Glycine)
what is the spliceosome
An RNA-protein complex of around 100 proteins and 5 small RNAs
to get from pre-mRNA to spliced mRNA.
Works similarly in different organisms, most complex in animals.
what are snRNP and non snRNP splicing proteins and their functions
what is the spliceosome cycle
how does splicing catalysis happen
what is the minor ATAC spliceosome
how can splicing occur in trans
Splicing occurs in trans in some parasites
2 mRNAs from different loci on the chromosome come together and undergo splicing.
Still uses GU-AG but the AG comes from an entirely different mRNA.
Uses the same factors, only difference = occurs in trans.
characteristics of alternative splicing
Mammals have evolved to use alternative splicing
Major source of isoform diversity
90% of pre-mRNAs undergo alternative splicing with ≥ 2 isoforms.
Functions: related, distinct, antagonistic functions and can be differentially localized.
always regulated with splice enhancers and splice inhibitors.
Serves as a post-transcriptional on/off switch for gene expression:
May lead to frameshifting: resulting in truncated proteins or Nonsense-mediated decay
Nuclear retention of unspliced RNA.
what is the metazoan gene structure
Usual composition = small exons + large introns
80-90% intron
what is cis splicing and what results can we get from it
what are some things that can go wrong with splicing
Things can go wrong:
Inactivate authentic splice sites.
Create ectopic splice sites in introns or exons.
Activate cryptic splice sites in introns or exons.
Create or destroy euxinic or intronic splicing enhancers.
Create or destroy exonic or intronic splicing silencers.
First splicing mutation was discovered in globin.
splicing and genetic diseases
spinal muscular atrophy, splicing and treatment
