#7 mRNA Processing

Pre-mRNA processing

co-transcriptional process, involves phosphorylation of CTD of RNAPolII to bind pre-mRNA processing factors, removal of introns (occurs very quick after transcription)

Consensus sequences

conserved mRNA sequences around 5'/3' splice sites

5' SS

5' splice site (donor site): GU dinucleotide

3' SS

3' splice site (acceptor site): AG dinucleotide

Polymerpyrimidine tract

upstream of 3' splice site

Branch point A

Branch point A: adenine used as a branch point in splicing reaction

Spliceosome

snRNPs, SR proteins and hnRNP proteins, removes introns from pre-mRNA

snRNP

5 distinct snRNAs with snRNP-specific proteins

SR proteins

RNA binding proteins, auxiliary factors, Ser/Arg dipeptide domains

hnRNP proteins

RNA binding proteins, package nascent mRNA

Introns

non-coding regions, very long in human genes, used to define borders for splicing

Alternative splicing

regulated process of including or excluding varying regions of pre-mRNA in the final mRNA, creates protein diversity but also genetic diseases, regulated by trans-acting proteins

Trans-acting proteins

repressors/activators that regulate alternative splicing by binding cis-acting RNA elements

Cis-acting RNA elements

silencers/enhancers elements of RNA that influence how splicing will occur

Postive regulation

cis-acting enhancing elements bind trans-acting factors

Purine-rich splicing enhancers

cis-acting element, mostly in exons, binding site for trans-acting factors (SR-proteins), often activate splicing of upstream intron,

SR proteins

Trans-acting splicing factor, POSTIVE regualtors of splicing, recognize exonic splicing enhancers (ESE) to activate suboptimal splice sites, required for constitutive splicing

RRM

RNA recognition motif domains, determins RNA binding specificity

RS domains

rich in alternating Arg/Ser residues, mediates protein-protein interactions to recruit splicing machinery

Pyrimidine-rich splicing enhancers

cis acting splicing enhancers, often in introns close to 5' splice site, help to recruit U1 snRNP to 5' splice site

Enhancer-dependant Spliceosome assembly

SR protein binds enhancer -> recruit U2AF (Splicing factor) -> recruits U2 snRNP

Negative regulation

occurs by factors that bind regulatory elements (ESS, ISS) within introns/exons (ex. hnRNP) or inhibitory secondary structure of pre-mRNA

-ESS: exonic splicing silencers

-ISS: intronic splicing silencers

Drosophila

sex in flies is determined by number of X chromosomes (2X female, X male), Sex-lethal protein coordinates sex determination/dosage compensation in females

Sex-lethal (sxl)

expressed in females with 2X chromosomes, early Sxl protein is transcribed in early embryogenesis, controls Tra protein splicing and inhibits Msl-2

Early sxl protein

sequence-specific RNA-binding protein that binds it's own pre-mRNA to form either Late Sxl protein or pre-mature stop codon (non-functional Sxl protein)

Msl-2

needed for dosage compensation in male flies that is inhibited by Sxl protein in females

Tra1 protein

in females, forms complex with TRA2 protein (RNA-binding SR protein), Sxl blocks splicing of exon 2/exon3 that contains stop codon

Tra1/Tra2 complex

activates upstream 3' splice site of doublesex gene in female flies, expressed as DSX(m) in male flies

DSX

in male or female splice isoforms based on Tra1 expression, transcriptional regulators required for sexual development

Alternative splicing in vertebrate cells

1. Variations in relative concentration/activity of splicing factors

2. Strength/arrangement of binding sites for regulators + constitutive splicing factors 

hnRNP A1

Negative regulator, antagonist of SR proteins, often exonic splicing repressor

hnRNP F/H

negative regulator, bind exonic splicing silencers

PTB (hnRNP 1)

negative regulator, intronic splicing repressor, often blocks poly-pyrimiding tract

Different protmoters

can influence transcription on splice/site choice by:

a) recruiting splicing factors (ex. SR) b) kinetics of transcription elongation

Chromatin configuration

can influence splice site by a) recuriting of splicing repressor or b) transcription elongation rate

Polyadenylation

addition of multiple alanines at end of mRNA, helps stimulate transcription termination, facilitates splicing of most 3' intron, generates different mRNAs, supoprts mRNA stability/export, translation initiation??

Coupled 2-step reaction of cutting + Polyadenylation

Poly (A) signal

combo of cis acting elements: AAUAA (conserved), CA PolyA site, U/GU rich downstream element, upstream element (+ or -)

Alternative polyadenylation

increases mRNA stability/translation/nuclear export/localization, protein localization

Non-polyadenylated transcripts

will be degrades or not transported efficiently into cytoplasm, decrease in protein expression

UTR-APA

Tandem arrays of polyA sites within single untranslated region. Helps process if 2 polyA sites

UR-APA

alternative polyadenylation in upstream regions, can produce isoforms with different 3'-terminal exons (aka different coding sequences and 3' UTRs) . Helps process if 2 polyA sites

Efficiency of processing complex assembly

regulated polyA sites are weak, effect of concentration of polyadenlation factrs, stabilization via accessory factors, tissue specific differences in RNA-binding proteins for polY site recognition (Mechanisms of Regulation)

Processing enzyme activity

regulated by post-translational modifications or repressor proteins (Mechanisms of Regulation)

IgM

secreted vs membrane-bound IgM (C-terminal tail has transmembrane anchor), regulated by Cst-64

Cst-64

high affinity for 2nd polyA site (membrane), in plasma cells, increasing Cst-64 concentration allows for weaker polyA site (secreted)