Modulating alternative splicing of heterogenous nuclear ribonuclear protein A2/B1 (hnRNP A2/B1) in HeLa cells




Manasa Venkataramani

Introduction

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.

The hnRNP family consists of 20 major polypeptides. hnRNP A2 and B1 proteins are two such members that are encoded by the gene HNRNPA2B1 (Kozu, Henrich, and Schäfer 1995, 2), that can produce either proteins by alternative splicing (Fig 1A). Naturally occurring compounds have been shown to alter splicing patterns by regulating the content of splicing factors (Anderson, Qiu, and Rubin 2003). The aim of this study was to modulate alternative splicing of the primary transcript from the gene HNRNPA2B1 using such compounds and examine if alteration in hnRNP A2/B1 splicing is reflected in the splicing of ay known targets of A2/B1 proteins.


Materials and Methods

HeLa cells were treated with various compounds for 48 hours. Cells were lysed and total cellular RNA was extracted using QIAGEN RNeasy Plus Mini Kit.
RT-PCR was performed using primers specific to amplify hnRNP A2/B1, ARAF and CASP9 transcripts, in RNA from treated and untreated cells using QIAGEN One-Step RT-PCR Kit.
Products of this amplification were analyzed on a 1% agarose gel, purified and sent out for Sanger sequencing.
Densitometric analysis was done using ImageJ software.


Results

RT-PCR in extracted RNA samples was performed using primers designed to anneal specifically to exons 1 and 3 of hnRNP A2/B1. Fig 1A shows that treatment 7 facilitated greater production of hnRNP B1 transcript relative to hnRNP A2. ImageJ analysis revealed that there was a two-fold increase in the ratio of B1/A2 transcript levels in treated cells when compared to that of RNA from untreated cells (Fig 1C).

ARAF gene encodes for a protein kinase A-Raf involved in the transduction of mitogenic signals, whose full-length isoform is oncogenic while the short, dominant-negative isoform acts as a tumor suppressor which is from the transcript in which introns 2 and 4 are included (Fig 2A). Studies indicated that hnRNP A2 favors the full length transcript of A-RAF while isoform B1 favors the shorter isoform (Shilo et al. 2014). RT-PCR in RNA from treated and untreated cell lysates was performed using primers designed to recognize sequences in exons 2 and 4 of ARAF to recognize both the transcripts. There was no difference in the expression of the intron-2 containing transcript upon treatment (Fig 2B).

CASP9, via alternative splicing generates two transcript variants: CASP9α in which all the exons are included and upon translation, results in a pro-apoptotic protein; CASP9β in which exons 3 through 6 are excluded, and gives rise to a shorter, anti-apoptotic protein (Srinivasula et al. 1999) (Fig 3A). Previous studies show that hnRNP-A2 over-expression favored the production of CASP9β transcript (Golan-Gerstl et al. 2011). RT-PCR in RNA from treated and untreated cell lysates was performed using primers designed to recognize sequences in exons 2 and 7 of CASP9. Treatment 7 increased CASP9α/CASP9β transcript levels (Fig 3B) and quantification revealed that the increase in the CASP9 α/β ratio was 40% upon treatment (Fig 3C).


Discussion

HNRNPA2B1 generates proteins A2 and B1, via alternative splicing. Treating HeLa cells with compound 7 increased the ratio of B1/A2 transcripts compared to that of untreated cells. To determine whether or not there was a change in individual transcripts, qRT-PCR specific to each transcript must be performed.

ARAF encodes a functional full-length A-Raf protein which acts an oncogene and a truncated, dominant negative isoform which acts as a tumor suppressor. Treatment 7 did not increase the expression of the intron-containing transcript. This might be because the increase in B1 transcript by treatment 7 was not sufficient to influence ARAF splicing. CASP9 encodes for pro-apoptotic Caspase-9α and anti-apoptotic Caspase-9β. HeLa, being a cervical cancer cell line had increased amounts of CASP9β (anti-apoptotic) and lesser CASP9α (pro-apoptotic) transcript levels, as expected. Treatment 7 increased CASP9α/β ratio. Given that CASP9 is an A2/B1 target, this change in CASP9 splicing may be attributed to the changes observed in hnRNP A2/B1 splicing, however it is also possible that the compound influences CASP9 splicing directly or indirectly by influencing the expression of some other gene.

To determine if the change in CASP9 splicing is due to altered A2/B1 expression, B1 must be overexpressed using a suitable expression vector to determine if the same effect is seen. Additionally hnRNP A2 should be silenced to determine its impact on CASP9 splicing.

This study shows that naturally occurring compounds have the ability to alter the splicing of hnRNP A2/B1. hnRNP A2/B1 proteins regulate the splicing of several oncogenes and tumor-suppressors. Exploring how naturally occurring compounds can alter the splicing of tumor-specific variants could provide insight into novel therapeutic approaches.


References

Anderson, Sylvia L., Jinsong Qiu, and Berish Y. Rubin. 2003. “EGCG Corrects Aberrant Splicing of IKAP MRNA in Cells from Patients with Familial Dysautonomia.” Biochemical and Biophysical Research Communications 310 (2): 627–33.

Golan-Gerstl, Regina, Michal Cohen, Asaf Shilo, Sung-Suk Suh, Arianna Bakàcs, Luigi Coppola, and Rotem Karni. 2011. “Splicing Factor HnRNP A2/B1 Regulates Tumor Suppressor Gene Splicing and Is an Oncogenic Driver in Glioblastoma.” Cancer Research 71 (13): 4464–72. https://doi.org/10.1158/0008-5472.CAN-10-4410.

Kozu, T., B. Henrich, and K. P. Schäfer. 1995. “Structure and Expression of the Gene (HNRPA2B1) Encoding the Human HnRNP Protein A2/B1.” Genomics 25 (2): 365–71.

Lee, Yeon, and Donald C. Rio. 2015. “Mechanisms and Regulation of Alternative Pre-MRNA Splicing.” Annual Review of Biochemistry 84: 291–323. https://doi.org/10.1146/annurev-biochem-060614-034316.

Shilo, Asaf, Vered Ben Hur, Polina Denichenko, Ilan Stein, Eli Pikarsky, Jens Rauch, Walter Kolch, Lars Zender, and Rotem Karni. 2014. “Splicing Factor HnRNP A2 Activates the Ras-MAPK-ERK Pathway by Controlling A-Raf Splicing in Hepatocellular Carcinoma Development.” RNA (New York, N.Y.) 20 (4): 505–15. https://doi.org/10.1261/rna.042259.113.

Srinivasula, S. M., M. Ahmad, Y. Guo, Y. Zhan, Y. Lazebnik, T. Fernandes-Alnemri, and E. S. Alnemri. 1999. “Identification of an Endogenous Dominant-Negative Short Isoform of Caspase-9 That Can Regulate Apoptosis.” Cancer Research 59 (5): 999–1002.

Figures


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.

Full Paper

Acknowledgments

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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