4. Mrna Splicing

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is a process where exons are ligated and the introns are removed.

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Constitutive splicing

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on the pre-mRNA are required to signal splicing

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Consensus sequences

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•5'splice site:
•3' splice site:
•Branch point:
•Pyrimidine tract:

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•AG↓GUAA
•CAG↓G
•(C/U)U(A/G)AC
•C/U

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RNA Splicing Mechanism

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“A series of transesterification reactions”

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Spliceosome Components

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Trans-factors – around 200 RNA binding proteins are transiently associated with the spliceosome.
•snRNAs
•snRNPs

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Small nuclear RNA (snRNAs) are complexed with special proteins and are therefore referred to as small nuclear RiboNucleoprotein Particles or snRNPs (snurps). Example?

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U1, role: binds the 5'splice site and then the 3' splice site.

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joins the 5 and 3 splice sites that define the exon-intron boundary and removes introns.

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The spliceosome (a large multicomponent ribonucleoprotein complex)

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Genes are capable of coding for more than 1 protein by the process of

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alternative splicing.

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Modes of Alternative RNA processing

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Selective splicing
Exon skipping
Alternate 5' splice site
Alternate 3' splice site
Alternate poly(A) site

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For efficient splicing, most introns require

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cis elements comprising of a conserved 5 splice site (AG↓GUpu), a branch point (BP) sequence (CupuApy) followed by a polypyrimidine tract and a 3 splice site (pyAG↓puN).

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Additional sequences exist in the pre-mRNA as auxiliary cis-elements that recruit

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trans-acting factors to promote splicing.

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The regulation of alternative splicing is often the result of

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dynamic antagonism between trans-acting proteins binding to these cis-elements.

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Auxiliary cis-elements can promote or repress

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splice site usage

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Depending on their location and their effect on recognition of alternative splice sites, these elements are referred to as

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exonic splicing enhancers (ESEs) or silencers (ESSs) and intronic splicing enhancers (ISEs) or silencers (ISSs)

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______are splicing trans-factors that bind to RNA and play essential roles in splicing.
and are shown to modulate alternative splicing by affecting the usage of competing 3' or 5' splice sites

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SR Proteins

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The serines in the RS-domain are highly phosphorylated.

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SR Proteins

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Splicing factors can also be regulated via

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signal transduction pathways.

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are splicing trans-factors that bind to RNA and play essential roles in splicing.
•are shown to repress exon splicing.
•hnRNPs may interfere with binding of other trans-factors to their cis-elements thereby suppressing that exon usage or splice site utilization.

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hnRNPs

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is a means Of expanding the coding power of the genome:

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Alternative splicing of pre-mRNA

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Alternative patterns of RNA splicing result from

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tissue-specific adaptive, developmental, or hormonal control mechanisms.

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Regulation of alternative splicing

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•Tissue-specific expression
•Developmental splicing
•Hormonal splicing

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Tissue-specific splicing example:

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making of tropomyosin family proteins.

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Developmental splicing example:

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V25 alternative variant, was significantly higher in all fetal

decrease in the EIIIA variant in brain

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Hormone-regulated splicing example:

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Insulin regulates alternative splicing
Of PKCßII mRNA via exon inclusion

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Mutations in the pre-mRNA causing NMD

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Marfan Syndrome

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Mutations in regulatory sequences

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•Spinal muscular atrophy

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faulty Exon usage, disease related:

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Frontotemporal dementia with parkinsonism linked to chromosome 17

•Breast and ovarian cancer

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Introduction of cryptic 5’ or 3’ splice sites

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Hutchinson-Gilford progeria syndrome (HGPS)– growth retardation and premature aging.

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Mutations affecting multiple functions of splicing

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Breast and ovarian cancer

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Aberrant splicing caused by disease states

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Diabetes and various cancers
Many mutations are silent until an external stimulus activates it
Atherosclerosis influences

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generates protein diversity.

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Alternative splicing

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Regulation of alternative splicing is achieved by

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binding of protein trans-factors to their cis-elements on the pre-mRNA.

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can be a cause or consequence of human disease.

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Changes in alternative splicing