Alternative splicing : mechanism and regulation

Alternative splicing is a deviation from the conventional splicing as it removes introns in a different manner. It has a lot of significance in the development of diseases like cancers and in plants adapting to various stress conditions.

1. WELCOME

2. ALTERNATIVE SPLICING : MECHANISM AND REGULATION SPEAKER SMRUTIREKHA SAHU ROLL NO. 20950 M.Sc. 1 st year DIVISION OF BIOCHEMISTRY CHAIRPERSON DR. ARCHANA SACHDEV PRINCIPAL SCIENTIST DIVION OF BIOCHEMISTRY CREDIT SEMINAR

3. ONE GENE MANY PROTEINS - ALTERNATIVE SPLICING

4. EUKARYOTIC GENE STRUCTURE

5. SPLICING Splicing is the editing of the nascent precursor messenger RNA (pre-mRNA) transcript into a mature messenger RNA (mRNA). After splicing, introns are removed and exons are joined together (ligated). For nuclear-encoded genes, splicing takes place within the nucleus either during or immediately after transcription. For those eukaryotic genes that contain introns, splicing is usually required in order to create an mRNA molecule that can be translated into protein. For many eukaryotic introns, splicing is carried out in a series of reactions which are catalysed by the spliceosome, a complex of snRNPs. Self-splicing introns, or ribozymes capable of catalysing their own excision from their parent RNA molecule, also exist.

6. SPLICING

7. The processing of bulk of eukaryotic mRNAs is mediated by particles called spliceosome , a large molecular complex that recognizes sequences in the pre-mRNA, called splice sites, to remove the noncoding introns and join the flanking exons. They are ellipsoid particles of RNA and protein (a ribonucleoprotein) and is ~25 X 50 nm in size. The spliceosome core consists of five small nuclear ribonucleoproteins (snRNP1, snRNP2, snRNP4, snRNP6, snRNP5) and numerous spliceosome-associated factors or proteins which assemble at introns in a precise order. SPLICEOSOME

8. SPLICEOSOME MEDIATED SPLICING PROCESS

9. HISTORY OF ALTERNATIVE SPLICING First discovered in 1977 in a bacteriophage ( adenovirus ) proposed by Gilbert. The thyroid hormone calcitonin was found to be alternatively spliced in mammalian cells. The primary transcript from this gene contains 6 exons; the calcitonin mRNA contains exons 1–4, and terminates after a polyadenylation site in exon 4. Another mRNA is produced from this pre-mRNA by skipping exon 4, and includes exons 1–3, 5, and 6. It encodes a protein known as CGRP (calcitonin gene related peptide). The "record-holder" for alternative splicing is a D. melanogaster gene called Dscam, which could potentially have 38,016 splice variants.

10. ALTERNATIVE SPLICING It is deviated from constitutive splicing which is a regulated process during gene expression that results in a single gene coding for multiple proteins wherein, particular exons maybe included or excluded from final, processed mRNA. Consequently, the proteins translated from alternatively spliced mRNAs will contain differences in their amino acid sequence and, often, in their biological functions. Alternative splicing occurs as a normal phenomenon in eukaryotes, where it greatly increases the biodiversity of proteins that can be encoded by the genome.

11. ALTERNATIVE SPLICING

12. ALTERNATIVE SPLICING PRODUCES 3 ISOFORMS OF PROTEIN

13. MODES OF ALTERNATIVE SPLICING Exon skipping or cassette exon : In this case, an exon may be spliced out of the primary transcript or retained. This is the most common mode in mammalian pre-mRNAs Mutually exclusive exons : One of two exons is retained in mRNAs after splicing, but not both.

14. Alternative donor site : An alternative 5' splice junction (donor site) is used, changing the 3' boundary of the upstream exon. Alternative acceptor site : An alternative 3' splice junction (acceptor site) is used, changing the 5' boundary of the downstream exon. Intron retention : A sequence may be spliced out as an intron or simply retained. This is distinguished from exon skipping because the retained sequence is not flanked by introns. If the retained intron is in the coding region, the intron must encode amino acids in frame with the neighbouring exons, or a stop codon or a shift in the reading frame will cause the protein to be non-functional. This is the rarest mode in mammals.

15. MECHANISM OF ALTERNATIVE SPLICING

16. SPLICE SITE SELECTION The decision to include and remove exons involves RNA sequence elements and trans acting factors such as : SR proteins hnRNPs RbFOX proteins Depending on the position and function of the cis-regulatory elements, they are divided into 4 categories: ESEs(Exonic splicing enhancers) ESSs(Exonic splicing silencers) ISEs(Intronic splicing enhancers) ISSs(Intronic splicing silencers)

17. They are a family of nuclear factors functioning in both constitutive and alternative RNA splicing. They bind to Exonic splicing enhancers(ESEs) through their RNA recognition motifs (RRMs) and mediating protein-protein, protein-RNA interactions through their RS (Arg-Ser repeat containing domains). Structure- one or two ribonuleoprotein particle type RNA binding domains at their amino termini and a variable length domain enriched in Arg-Ser dipeptides at their carboxyl termini (RS domain) RS domains are phosphorylated and act as activators of splicing. SR PROTEINS

18. U1 snRNP U2 snRNP U1 snRNP U1 snRNP U1 snRNP U1 snRNP U1 snRNP U1 snRNP U2 snRNP U2 snRNP U2 snRNP U2 snRNP U2 snRNP U2 snRNP

19. The Rbfox proteins all contain a single highly conserved RNA recognition motif (RRM) that specifically binds the sequences UGCAUG and GCAU The binding of Rbfox to a (U)GCAUG element downstream of the alternative exon promotes its splicing, whereas binding to an upstream element, or an element within the exon, represses exon inclusion. RB FOX PROTEINS

20. Heterogeneous nuclear ribonucleoproteins (hnRNPs) comprise a family of RNA-binding proteins. They are multifunctional, involved not only in processing heterogeneous nuclear RNAs (hnRNAs) into mature mRNAs, but also acting as trans -factors in regulating gene expression. Heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1), a subgroup of hnRNPs, is a KH-triple repeat containing RNA-binding protein. It is encoded by an intronless gene arising from hnRNP E2 through a retrotransposition event. The most straightforward mode of action is based on a competition between hnRNP proteins and other splicing factors for binding to cis -elements having a splicing regulatory function . Negative regulation can be achieved by occlusion of 5′ or 3′ splice sites, branch point and polypyrimidine tract, or binding sites of activating SR proteins hnRNP PROTEINS

21. RNA-RNA BASE PAIRING RNA-RNA and RNA-protein interactions that regulate mutually exclusive splicing of Drosophila DSCAM exon 6 cluster

22. REGULATION

23. Two models have been proposed to explain the role of RNAP II in the regulation of alternative splicing. THE RECRUITMENT MODEL RNAP II and transcription factors interact, directly or indirectly, with splicing factors, thereby increasing or decreasing the efficiency of splicing THE KINETIC MODEL It proposes that the rate of transcription elongation influences the inclusion of alternative exons by affecting whether the splicing machinery is recruited sufficiently quickly for spliceosome assembly and splicing to occur REGULATION BY TRANSCRIPTION COUPLING

24. During RNA polymerase II (Pol II)-mediated transcription, fast elongation (left) favours the recruitment of the spliceosome to the strong 3′ splice site of a downstream intron instead of the weak 3′ splice site of the upstream intron, which results in exon skipping. By contrast, slow elongation (right) favours the recruitment of spliceosome components to the upstream intron, which results in splicing commitment and exon inclusion In conditions in which both 3′ splice sites are equally strong and the upstream intron also has a binding site (green) for a splicing factor that inhibits exon inclusion (negative factor (NF)), fast elongation of Pol II (left) favours the recruitment of spliceosome components to both introns, ensuring exon definition and subsequent exon inclusion. On the contrary, slow elongation (right) provides a time window for the negative splicing factor to be recruited to its target site before spliceosome components can bind to the 5′ splice site of the downstream intron and mediate exon definition. This results in exon skipping.

25. Adapters that link specific histone modifications or marks to splicing factors H3K9me2 and H3K9me3 are recognized and bound by HP1α and HP1γ, respectively. As a result, Pol II slows down at the HP1-bound chromosomal region, leading to increased inclusion of a nearby alternative exon. Hyperacetylation of H3 and H4 promotes a more relaxed chromatin structure, increased Pol II elongation rate and skipping of alternative exons REGULATION BY CHROMATIN STRUCTURE

26. The chromodomain protein MRG15 binds to H3K36me3 and recruits the splicing silencer protein PTB to its target RNA, thereby promoting skipping of an alternative exon. Another H3K36me3-binding protein, Psip1, affects inclusion of alternative exons by recruiting the splicing regulator SRSF1.

27. MODEL OF ALTERNATIVE SPLICING REGULATION BY DNA METHYLATION AND CTCF ACCUMULATION DESCRIBED FOR EXON 5 (E5) OF THE CD45 GENE E5 skipping is favoured by fast RNA polymerase II (RNAPII) elongation rates promoted by specific DNA methylation in the exonic region that inhibits CTCF binding. E4 and E6 skipping, on the other hand, is promoted by hnRNPL binding to pre-mRNA. In the absence of DNA methylation, CTCF binds to E5 DNA where it creates roadblocks to RNAPII elongation favouring E5 recognition and inclusion. E4 and E6 skipping is not affected by this mechanism.

28. REGULATION BY ATIs AND ATTs These occur in the UTRs as compared to other splicing events that takes part within the coding sequences. This reflects the potential regulation of large distinct groups of genes with different mechanisms, such as strong coupling with alternative splicing in 5’ and 3’ UTRs.

29. ALTERNATIVE SPLICING AND NMD EXON JUNCTION COMPLEX

30. ALTERNATIVE SPLICING AND DISEASES

31. Many of these diseases arise from multiple distinct molecular mechanisms. The exon-specific detection of alternative splicing might serve as a reliable biomarker and provide a novel approach to diagnose and monitor disease progression ALTERNATIVE SPLICING AND DRUG DESIGNING

32. Exitrons are internal parts of protein-coding exons that are hidden in the exonic sequence and that are alternatively spliced. They combine features of both exons and introns ALTERNATIVE SPLICING AND DECODING GENE EVOLUTION

33. ENVIRONMENTAL STRESSES Functional full length proteins NMD insensitive mRNAs Splicing machinery Splicing machinery Stable truncated protein Sequestered intron retaining mRNAs PTC+ unproductive mRNA Functional fully spliced mRNAs Daily transcriptional oscillations of pre mRNA -------------------------------------------------------------------------------- ----------------------------------- ---------------- -------------------------- Stable oscillations of functional mRNA Splicing of retained introns, translation ^ ^ Arrested splicing storage Possible translation of truncated proteins Feedback to the pool of functional mRNA constitutive splicing alternative unproductive splicing Transcript degradation NMD eliciting mRNAs ALTERNATIVE SPLICING AND STRESSES

34. EQUILLIBRIUM OF FUNCTIONAL AND PTC+ mRNAs UNDER NORMAL CONDITIONS INCREASE OF FUNCTIONAL ISOFORM INCREASE OF PTC+ ISOFORMS ENVIRONMENTAL STRESS B ENVIRONMENTAL STRESS A CONTD.

35. ABA SIGNALLING AND ALTERNATIVE SPLICING

36. INTRODUCTION Due to the sessile lifestyle, plants face fluctuating environmental conditions. In order to both benefit maximally and to protect themselves from environment, plants evolved ways to sense and responds to many environmental cues. Ambient temperature is one of these signals that plants sense and adapt to in order to enhance their chance of survival and reproduction where small changes could have major effects on plant architecture and development. One of them being the moment of flowering. Environmental changes trigger differential AS. An intron containing gene can potentially produce several to numerous different splice forms by combining conventional splicing with alternative selection of splice sites such as Retention of introns (RI), skipping or mutual exclusion of exons (SE or MSE), alternative splice site selection at 5` or 3 ` end (A5 or A3).

37. STUDY They analysed ambient temperature-directed AS in two accessions of A. thaliana and one accession of cauliflower (B. oleracea ssp. botrytis). Analysis of a mutant of a splicing-related gene in A. thaliana Col-0 ( SALK_144790), for which AS was observed in this accession and its orthologous gene in cauliflower, showed an altered flowering time response under different ambient temperatures. This suggest that AS of splicing-related genes functions as a key molecular mechanism in plant’s temperature response.

38. MATERIALS AND METHODS thaliana Col-0 and Gy-0 seeds were sown stratified for 2-3 days at 4ºC transferred to 23ºC for 3 weeks(in case of high temperature treatment) or 6 weeks(in case of low temperature treatment) vegetative state -- transferred to 27˚C (Col-0 and Gy-0) or 16˚C (Col-0) for 24 hours and then above ground parts were harvested Brassica oleracea var. botrytis F1 seeds are sown transferred to the greenhouse under long day conditions (day: 16 hours at 21˚C/night: 8 hours at 16˚C) for 2 weeks after five weeks, half of the plants were transferred to higher ambient temperature (day/night: 27˚C /22˚C) after 24 hours, meristem-enriched tissue was harvested

39. RESULTS The effect of ambient temperature on alternative splicing: FIG1. A. OVERVIEW OF THE SPLICING EVENTS THAT CAN OCCUR B. DISTRIBUTION OF DIFFERENTIAL SPLICING EVENTS UPON SHIFTS TO HIGHER OR LOWER AMBIENT TEMPERATURE

40. RESULTS A.(TOP)RAW READS FROM IGV (BOTTOM)INTRON EXON STRUCTURE WITH ALL DETECTED SPLICING EVENTS B. (TOP)ALTERNATIVE SPLICING UPON LOW AMBIENT TEMPERATURE IN THE IGV (BOTTOM)THE SKIPPING OF EXON 2 IS DIFFERENTIAL INDICATED BY * C. RT-PCR FOUR DIFFERENT SPLICING EVENTS RESULT IN 6 DIFFERENT SPLICE FORMS FIG.2

41. Splicing related genes are overexpressed: FIG. 3 GO TERM ENRICHMENT OF DIFFERENTIALLY SPLICED ARABISDOPSIS Col-0 GENES UPON AMBIENT TEMPEARTURE TREATMENT

42. The spliceosome is the target of ambient temperature-induced alternative splicing: FIG 4.CLASSES OF SPLICING RELATED ARABIDOPSIS Col-0 GENES THAT SHOWS DIFFERENTIAL SPLICING OF ATLEAST ONE GENES

43. . FIG 5. Flowering time analysis of A. thaliana Gy-0. Plants were grown under 22°C and 27°C. Flowering time was determined by counting rosette leaves at the moment of flower induction. N=10, mean ±SD Alternative splicing of splicing related genes in other genetic backgrounds:

44. A role for the differentially spliced splicing-related gene ATU2AF65A in thermosensitive floral timing: Gene structure of ATU2AF65A showing the position of TDNA insertion in SALK_144790 Wild type and mutant have the same phenotype at 16ºc The mutant showed an increased flowering time response upon higher ambient temperatue

45. Their study unveiled that, upon small temperature changes, spliceosomal genes are overrepresented amongst the differentially spliced genes, and moreover, that this included many classes of splicing related genes thus showing that The splicing machinery was a target for regulation. The mutant for ATU2AF65A gene did not show any significant differences in expression suggested a biological function for the ambient temperature directed splicing of ATU2AF65A Temperature sensing through alternative splicing: A two-step model CONCLUSION

46. CONCLUSION FOR THE SEMINAR Regulation of alternative splicing represents an important means to fine-tune gene expression that saves time required for changes in transcriptional activation and pre-mRNA accumulation, thus allowing rapid plant adaptation to adverse environmental stresses. Individual splicing regulators control much larger group of genes than specific transcription factors. The combination of alternative splicing database, tandem mass spectrometry may aid with identification, analysis and characterization of potential alternative splicing isoforms. Combining alternative splice variants dramatically expands the proteomics of genomes.