RNA Splicing is catalyzed by a set of proteins collectively called the spliceosome. These proteins bind 5’ and 3’ splice sites at the intron-exon junctions, leading to the removal of introns. The information regarding splice site usage and specificity lies in cis- regulatory elements (CREs) which have the ability to recruit trans- acting splicing factors (Lee and Rio 2015). Serine/Arginine-rich (SR) proteins and heterogenous nuclear ribonuclear proteins (hnRNPs) are the most common families of splicing factors. These bind to the CREs and facilitate or inhibit the use of splice sites, resulting in alternative splicing.
Figure 1-(A) Schematic of alternative splicing in HNRNPA2B1 primary transcript, insertion of exon 2 results in hnRNP B1 protein while its exclusion leads to the production of hnRNP A2 protein. (B) RT-PCR using primers amplifying both the transcripts of hnRNP A2/B1, arrows indicate the bands corresponding to the expected product sizes. The identity of the two isoforms were verified through sequencing (UT- untreated; 7- treatment #7; NTC- non-template control). (C) Graph showing the ratio of B1/A2 transcript levels in untreated and treated cells.
Figure 2-(A) Schematic of alternative splicing in ARAF primary transcript, inclusion of introns 2 and 4, generates a truncated protein due to the presence of a premature stop codon in intron 4 (red star).(B) RT-PCR using primers amplifying both the transcripts of ARAF gene, arrows indicate the bands corresponding to the expected product sizes. The identity of the of the transcript corresponding to Full-length (FL) protein was verified through sequencing (UT- untreated; 7- treated with compound #7; NTC- non-template control).
Figure 3-(A) Schematic of the splicing patterns in CASP9 primary transcript, skipping of exons 3 through 6 results in the generation of an anti-apoptotic, dominant-negative isoform of Caspase-9 protein. (B) RT-PCR using primers amplifying both the transcripts of CASP9 gene, arrows indicate the bands corresponding to the expected product sizes. The identity of the two isoforms were verified through sequencing (UT-untreated; 7- treated with compound #7). (C) Graph showing the ratio of CASPα/β transcript levels in untreated and treated cells.
The HNRNPA2B1 gene encodes for two proteins: heterogenous nuclear ribonuclear proteins A2 and B1, via alternative splicing. These act as splicing factors on several target genes and influence their splicing. In this study, HeLa cells were treated with different compounds that were known to alter splicing patterns. RT-PCR was performed using primers designed to amplify hnRNP A2 and B1 transcripts in RNA extracted from treated and untreated cells. The results showed that compound 7 altered splicing by facilitating the production of more hnRNP B1 relative to A2 transcripts. This prompted an examination of the splicing pattern of CASP9 and ARAF, known targets of A2/B1 proteins. ARAF generates many transcripts two of which are: the full length transcript with all the introns spliced out, generating an onco-protein and a transcript with introns 2 and 4 included encoding for a tumor-suppressor. RT-PCR in treated and untreated cells showed no difference in expression of these transcripts. CASP9 generates 2 transcripts CASP9α and β; CASP9β encodes an anti-apoptotic and oncogenic protein, while CASP9α encodes for a pro-apoptotic protein. To assess the impact of compound 7 on CASP9 splicing, RT-PCR using primers to amplify both α and β transcripts was performed in RNA isolated from cells treated with compound 7. The results indicated an increase in the ratio of CASP9α/β transcripts upon treatment. This change may be attributed to the increase in hnRNP B1/A2 ratio, but further studies need to be done to determine if alteration in CASP9 splicing was due to change in hnRNP A2/B1 splicing.
I would like to thank Dr. Rubin, for providing me the opportunity to work on this project and for his expert guidance which made this project feasible. I would also like to thank Anthony Evans and Devin Rocks for their enormous help and patience throughout the project. I am extremely grateful to Faaria Fasih-Ahmad for giving me her valuable suggestions in designing this project and providing me with treated HeLa cells.
|This document was last modified 05/14/2019.|
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