Alternative Splicing and Protein Isoforms

Alternative Splicing

• form of post-transcriptional, pre-mRNA processing in eukaryotes

• a gene can be spliced in different ways to produce variants of the same protein

• generates more diversity of proteins

• mutations occur at splice donor and acceptor sites, leading to exon skipping/intron retention → alternative splicing

• alternative splicing differs between species

Splice Sites

• special recognition sequences on the pre-mRNA are located at the intron-exon junctions and within the intron

  • required for intron removal

  • recognized by SnRNPs

• Splice donor site (5’ end of intron) → usually starts with GU

• Splice acceptor site (3’ end of intron) → usually ends with AG

Spliceosome

• ribonucleoprotein (RNP) complex found in eukaryotic nuclei that removes introns (non-coding regions) from pre-mRNA and joins exons (coding regions) to form mature mRNA

• built from five major small nuclear ribonucleoproteins (snRNPs): U1, U2, U4, U5, and U6

Alternative Splicing Products

• alternative splicing can lead to many alternative products and alternative expression patterns

• mutually exclusive alternative exons: only include one or the other

• alternative 5’/3’ splice site: within an exon, the sequence looks like a splice set and can get recognized as one

• alternative promoter and first exon: can have diff “first” exons with a gene that has diff promoters to drive expression

  • still result in transcription of the same gene, but w/ diff exons

• alternative poly A site and terminal exon: can have diff exons; exons have poly A sites

Constitutive Exon

• exon is always included in the transcript

Alternative Exon

• exon is not always included in the transcript

Weak consensus splice sites

• weak junctions: sequences don’t really look like splice sites

• might result in the exon getting skipped over along with the introns

Splicing Control Elements

• splicing control elements provide another level of regulation

• sequences within introns and exons that recruit factors can promote or suppress recruitment of the spliceosome to the splice sites (help or block spliceosome w/ binding or cleaving)

• analagous to TFs and enhancers that can promote or suppress recruitment of RNA pol to the promoter

• two components that regulate alternative splicing: cis-elements and trans-acting splicing factors

Cis-acting splicing regulatory elements

• defining an exon involves the specific stabilization or destabilization of splice site recognition

Enhancers

• stabilization: the splice site is recognized more often and the exon is included

• exonic splicing enhancers (ESE), intronic splicing enhancers (ISE)

Silencers

• destabilization: the splice site is suppressed and the exon is skipped

• exonic splicing silencers (ESS), intronic splicing silencers (ISS)

Classes of splicing regulatory elements figure

Alternative Splicing Regulation in Drosophila Sex: Sex-Lethal

• development of sex characteristics depends on the expression (or not) of a master regulator gene called sex-lethal (Sxl)

• functional Sxl protein is only produced if 2 X chromosomes are present

• XX:AA typically develop female phenotypes (Sxl is produced)

• XO:AA or XY:AA typically develop male phenotypes

Alternative Splicing Regulation in Drosophila Sex: mRNA splicing cascade

• in Drosophila, female and male biological phenotypes determined first by X dosage and then an mRNA splicing cascade

• functional Sxl protein initially expressed by an alternate promoter in XX embryos

• slightly later, Sxl transcribed in all embryos (XX, XO, XY) from a maintenance promoter

  • However, splicing outcome differs depending on whether Sxl protein is already present

• females: Functional Sxl protein is present → Sxl protein binds pre-mRNA and blocks inclusion of exon 3 → functional Sxl protein continues to be produced (positive feedback)

• males: No functional Sxl protein initially → exon 3 contains a premature stop codon → produces truncated, nonfunctional Sxl protein

• therefore Sxl must be a splice factor that recognizes an intronic splice silencer

Alternative Splicing Regulation in Drosophila Sex: Transformer

• functional Sxl protein only produced in XX (female) Drosophila

• functional Sxl regulates its own splicing as well as splicing of Transformer (Tra) transcripts

• functional Tra regulates splicing of Double-sex transcripts

• if Sxl and Tra are present: version of Dxs that regulates development of female genitalia

• if Sxl and Tra are absent, version of Dsx that regulates development of male genitalia

• Tra must be a splice factor that recognizes exonic splice enhancers (binds to exon 4 and promotes recognition of splice site

ISS in Transformer Transcript

• males: Sxl is absent; splicing factors recognize the splice acceptor site within intron 1, so exon 2 is included in the mRNA

  • exon 2 contains a premature stop codon, resulting in non functional Tra protein

• females: Sxl recognizes and binds to the ISS within intron 1, causing exon 2 and its premature stop codon to be spliced out of the mRNA

  • result: transcript for a functional Tra protein

ESE in Double-Sex Transcript

• the intron between exon 3 and 4 has only a weak consensus for binding the spliceosome complex, so exon 4 is skipped in males

• females: binding of Tra enhances spliceosome binding and promotes splicing between exon 3 and 4

  • exon 4 has its own polyadenylation signal, so exon 5 is excluded

How can you detect alternative splicing regulatory sequences and events? Large-scale genome-wide approaches

• RNA-seq (next gen-sequencing) to identify transcript variants and their relative amounts

• comparison of cDNA sequences to reference genome to ID exon-exon junctions (and introns)

  • ie. comparison with an existing exon-exon junction library

• DNA microarrays using exons instead of entire gene sequences

• cross-linking and immunoprecipitation: crosslink splicing factors and RNA transcripts, then use antibodies to immunoprecipitate the complexed RNA for sequencing

How can you detect alternative splicing regulatory sequences and events? Smaller-scale approaches

• RT-PCR and gel electrophoresis to detect alternate splice variants thru differences in size

  • Northern blots also possible

• EMSA: establish binding of spliceosome factors to RNA transcripts

• use of splicing reporter genes

Example of RT-PCR to detect presence or absence of splicing events: Ret-1

C. elegans RNA binding protein UNC-75 is responsible for splicing of neuronal transcripts

• N2: Wt worms

• unc-75: worms w/ lof mutation

• ret-1 is a transcript found in neurons. PCR primers span the indicated exons and introns

• from the data, we can say that UNC-75 protein is binding to an exonic/intronic splicing silencer

Example of RT-PCR to detect presence or absence of splicing events: C07A12.7 exon 4

C. elegans RNA binding protein UNC-75 is responsible for splicing of neuronal transcripts

• N2: Wt worms

• unc-75: worms w/ lof mutation

• C07A12.7 is a transcript found in neurons. PCR primers span the indicated exons and introns

• from the data, we can say the UNC-75 protein binds to an exonic/intronic splicing silencer

Strategies for pinpointing the regulatory elements bound by UNC-75 protein

• comparison of orthologous exons in other species of Caenorhabditis to find common sequence motifs. Consensus sequence: (G/U)UGUUGUG

• gel shift assay (EMSA) to esablish if UNC-75 can bind to this motif within these transcripts. Increasing the amounts of UNC-75 are added to the RNA

Bi-chromatic (two color) fluorescent splicing reporters

• tissue and temporal specificity of alternate isoforms can be investigated by generating reporter constructs expressing distinct fluorescent markers dependent on the expressed isoform

• this allows for studying alternate splicing in living cells

  • the color you get in the final protein depends on what exons are kept

Bi-chromatic (two color) fluorescent splicing reporters: Example

The ret-5 exon splicing reporter construct

• reporter construct contains reporter sequences for mCherry (red) and EGFP (green) in different frames

• neither reporter is expressed in the absence of splicing

• if exon 5 is skipped, mCherry sequences are in frame and cells turn red

• if exon 5 is retained, EGFP sequences are in frame and cells turn green