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17 Cards in this Set

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splicing- Transesterification

two transesterification reactions in pre-mRNA splicing

-2'OH group branch-site adenosine attacks the phosphate group at the 5'splice site intron

-transestification occurs

3'OH group at the 5' spice site attacks phosphate at 3' spice site- transesterification

intron removed known as branched lariat, converted to linear RNA by a debranching enzyme and is rapidly degraded by nuclear exosome

Consensus sequence

defines the splice sites in eukaryotic pre-mRNAs

The spliceosome

-what is it

-what does it consist of

A large ribonucleoprotein comples that mediates pre-mRNA slicing

-contains 5 small nuclear RNS (snRNAs)

U1, U2, U4, U5, U6 between 107-210 nts long

and contains ~170 proteins

snRNAs bind to consensus sequence on pre-mRNA to define spice-site selection

Spliceosome Mechanism

-U1 snRNP assembles at 5' splice site

-U2 snRNP assembles at 3' splice site

-Splicing factor 1 (SF1) assembles at branch point A

-A trimeric snRNP complex (U4, U5 and U6) joins to form the splicesome

-rearrangement of base-pairing interactions to form catalytically active spliceosome

-U1 andU4 snRNPs released

-catalytic core catalyses the first transesterification reaction- forms intron lariat

-Further rearrangement joins the two exons in a second transesterification reaction releasing the lariat intron as well as U2, U5 and U6

-The excised lariat intron is converted to linear RNA by a debranching enzyme and degraded by the exosome

Exons Vs introns



exons- 10 times shorter and more uniformed than introns

10 exons per gene

introns- 1.5KB long

largest 1.1 Mb long

Exons definition hypothesis

introns very long so additional factors (SR proteins) help to guide snRNPs to splice-sites

-serine-arginine-rich (SR) proteins preferentially bind to exon sequences in the pre-mRNA

-hnRNPs (heterogenous ribonucleoprotein particles may preferentially bind to intron sequences


-what is it?


-inherited blood disorder

-severe anemia due to aberrant (not normal) haemoglobin synthesis

-common in eastern mediteranean due to heterozygotes thought to be protected from malaria

-caused by mutations in the beta-globin gene

-causes portions of exons to be deleted and not transcribed

Alternative splicing

-forms different mRNA variants from the same gene

-may code for a different protein or have different mRNA properties eg stability

-therefore more mRNA variations than gene

Most extreme example of alternative RNA splicing

The drosophila DSCAM gene

can generate 38,016 possible isoforms from one gene!!!

Patterns of alternative splicing

-exon skipping

-intron retention

-alternative 5' splice site

-alternative 3' splice site

-mutually exclusive exons

α-tropomyosin- alternative splicing


coiled coil protein

regulates muscle contraction

alternative splicing cases variants of α-tropomyosin in different tissue

Control of alternative slicing by proteins

Activator proteins- bind to transcript and activate splicing that wouldn't happen otherwise

repressor proteins- binds to transcript inhibiting splicing by hindering access of the splicesome

RNA editing

changes in the nucleotide sequence of the mRNA

-insertion/deletion- of uridine (uracil nucleotide) in mitochondria of pathogenic trypanosomas (lower eukaryotes)

-substitution editing- by base deanimation- c to U

A to inosine (i)

C to U editing example

-C to U editing of APOB pre-mRNA

-Tissue-specific isoforms of apolipoprotein B, a protein required for uptake and transport of cholesterol eg c to u editing occurs in intestine but not liver to give different forms

•Site-specific deamination in the intestine changes the CAA codon (coding for Gln) to a UAA stop codon

•Reaction is catalysed by a cytidine deaminase that selectively recognise the RNA sequence around deamination site.

A to I editing example

A to I editing in pre-mRNAs of GluR-B

-GluR-B- a glutamate receptor subunit

-A to I editing can occur here in inotrophic glutamate recptor AMPA

-I is read as G during translation

-effects Ca2+ permeability of the receptor

-A to I deanimation done by 'adenosine deaminase acting on RNA' (ADARs)

RNA export

-Nuclear export factors (NXF1/T1) assemble with mRNA in the nucleus

-NXF/T are released in the cytoplasm and reimported to the nucleus

-Nuclear cap-binding protein (CBC) and PABPN1 are exchanged for cytoplasmic cap-binding protein (eIF4e) and PABPC1 in the cytoplasm

What drives the direction of mRNP export

-reversible phosphorylation of SR proteins