Alternative RNA splicing in cancer: what about adult T-cell leukemia?

Abstract

Eukaryotic cells employ a broad range of mechanisms to regulate gene expression. Among others, mRNA alternative splicing is a key process. It consists of introns removal from an immature mRNA (pre-mRNA) via a transesterification reaction to create a mature mRNA molecule. Large-scale genomic studies have shown that in the human genome, almost 95% of protein-encoding genes go through alternative splicing and produce transcripts with different exons combinations (and sometimes retained introns), thus increasing the proteome diversity. Considering the importance of RNA regulation in cellular proliferation, survival, and differentiation, alterations in the alternative splicing pathway have been linked to several human cancers, including adult T-cell leukemia/lymphoma (ATL). ATL is an aggressive and fatal malignancy caused by the Human T-cell leukemia virus type 1 (HTLV-1). HTLV-1 genome encodes for two oncoproteins: Tax and HBZ, both playing significant roles in the transformation of infected cells and ATL onset. Here, we review current knowledge on alternative splicing and its link to cancers and reflect on how dysregulation of this pathway could participate in HTLV-1-induced cellular transformation and adult T-cell leukemia/lymphoma development.

Keywords: Alternative splicing; Chemoresistance; HTLV-1; Leukemia; Oncogenesis.

Figure 1

Recapitulative diagram of HTLV-1 proviral genome, Open Reading Frames (ORFs), and splicing-regulated genes. HTLV-1 genome is 9 kb long and flanked on each side by 5’ and 3’ Long Terminal Repeats (LTRs). HTLV-1 LTRs are approximately 750 bp in length and are segmented in U3, R and U5 regions (in order). U3 region usually contains enhancer and promoter sequences which drive viral transcription; and R domain encodes the 5′ capping sequences (5′ cap) and the polyadenylation (pA) signal. The structural genes gag, pro, pol and env are encoded in the 5’ part of the provirus, and regulatory and auxiliary proteins are encoded in the pX region, at the 3’ side. The transcription initiates in the promoter-bearing 5’-LTR in 5’→3’ for all viral genes, except for the antisense transcripts of hbz. hbz is encoded by the proviral complementary strand and its transcription, initiated in the 3’-LTR, occurs in an antisense fashion (3’→5’). The different Open Reading Frames (ORFs) are indicated in roman numbers and splicing events are shown by dotted lines.

Figure 2

Step-by-step spliceosome assembly and pre-mRNA splicing reaction. Splicing is catalyzed by a large protein complex called the spliceosome. Spliceosome assembly requires a series of steps and intermediate complexes, and starts at the transcription site. It involves 5 small nuclear ribonucleoprotein particles (snRNPs): U1, U2, U4, U5 and U6; combined with roughly 300 associated proteins. Splicing is based on the recognition of 5’SS and 3’SS (splicing sites), also known as donor or acceptor sites, located at each end of an intron. Several cis-acting regulatory sequences are necessary such as the branching point sequence (BPS) and the poly-pyrimidine tract (PPT). Splicing begins with U1 snRNP recognition of the 5’SS and binding onto the pre-mRNA. U2 auxiliary factor (U2AF) 65 and 35kDa sub-units then respectively bind the PPT and the 3’SS; and Splicing Factor 1 (SF1) the BPS. These first steps form the E(arly) complex, which converts into pre-spliceosome complex A after U2 snRNP recruitment at the BPS and SF1 replacement. U2AF then leaves and U4, U5 and U6 pre-assemble into the tri-snRNP which is recruited to compose the pre-catalytic complex B. Rearrangement and catalytic activation into complex B^A occur via U1 and U4 release. A first trans-esterification reaction is catalyzed and leads to the Complex C containing free Exon 1 and Intron-Exon 2 fragment. The complex undergoes additional rearrangement and activation, and the Complex C^A catalyzes the second trans-esterification reaction to free Exon 2. Both exons are ligated in the Post-splicing Complex; U2, U5 and U6 as well as excised intron are released; and mature mRNA is formed. All snRNPs are recycled for additional rounds of splicing.

Figure 3

pre-mRNA alternative splicing events and regulation mechanisms. (A) Common constitutive and alternative splicing events are listed here. Colored boxes represent different exons, grey lines stand for introns, and dotted lines are splicing events. Exons are usually included or excluded (skipped) individually, but mutually exclusive exons involve the preferential retention of one exon at the expense of one or more others. Alternative 5’ and 3’SS selection induce exons modifications, as parts of the exons can be excluded during the process. Introns are mostly removed from the mRNA but inclusion can occur and often leads to nonsense-mediated decay or shift in the Open Reading Frame (ORF). Alternative promoters and poly (A) sites selection can also happen at the splicing level. All those events contribute to the increase of the proteome diversity. (B) Alternative splicing is regulated by trans-acting regulatory proteins, named splicing factors (SFs), binding to cis-acting regulatory sequences. SFs such as SR proteins and hnRNPs are RNA-binding proteins and are considered as enhancers and silencers respectively, since SR proteins are typically recruited to ISEs and ESEs while hnRNPs usually bind to ISSs and ESSs. However, increasing number of studies have revealed more context-dependent regulation roles for each. Other SFs are also at stake, and it is the balance and interplay between those splicing activators and repressors, and with the spliceosome components, that determines the splicing donor and acceptor sites selection for a splicing event to occur.