Jean-François Lucier

Cancer-associated regulation of alternative splicing.

Alternative splicing of pre-mRNA increases the diversity of protein functions. Here we show that about half of all active alternative splicing events in ovarian and breast tissues are changed in tumors, and many seem to be regulated by a single factor; sequence analysis revealed binding sites for the RNA binding protein FOX2 downstream of one-third of the exons skipped in cancer. High-resolution analysis of FOX2 binding sites defined the precise positions relative to alternative exons at which the protein may function as either a silencer or an enhancer. Most of the identified targets were shifted in the same direction by FOX2 depletion in cell lines as they were in breast and ovarian cancer tissues. Notably, we found expression of FOX2 itself is downregulated in ovarian cancer and its splicing is altered in breast cancer samples. These results suggest that the decreased expression of FOX2 in cancer tissues modulates splicing and controls proliferation.

Identification of Alternative Splicing Markers for Breast Cancer

Breast cancer is the most common cause of cancer death among women under age 50 years, so it is imperative to identify molecular markers to improve diagnosis and prognosis of this disease. Here, we present a new approach for the identification of breast cancer markers that does not measure gene expression but instead uses the ratio of alternatively spliced mRNAs as its indicator. Using a high-throughput reverse transcription-PCR-based system for splicing annotation, we monitored the alternative splicing profiles of 600 cancer-associated genes in a panel of 21 normal and 26 cancerous breast tissues. We validated 41 alternative splicing events that significantly differed in breast tumors relative to normal breast tissues. Most cancer-specific changes in splicing that disrupt known protein domains support an increase in cell proliferation or survival consistent with a functional role for alternative splicing in cancer. In a blind screen, a classifier based on the 12 best cancer-associated splicing events correctly identified cancer tissues with 96% accuracy. Moreover, a subset of these alternative splicing events could order tissues according to histopathologic grade, and 5 markers were validated in a further blind set of 19 grade 1 and 19 grade 3 tumor samples. These results provide a simple alternative for the classification of normal and cancerous breast tumor tissues and underscore the putative role of alternative splicing in the biology of cancer.

Multiple and Specific mRNA Processing Targets for the Major Human hnRNP Proteins

ABSTRACT Alternative splicing is a key mechanism regulating gene expression, and it is often used to produce antagonistic activities particularly in apoptotic genes. Heterogeneous nuclear ribonucleoparticle (hnRNP) proteins form a family of RNA-binding proteins that coat nascent pre-mRNAs. Many but not all major hnRNP proteins have been shown to participate in splicing control. The range and specificity of hnRNP protein action remain poorly documented, even for those affecting splice site selection. We used RNA interference and a reverse transcription-PCR screening platform to examine the implications of 14 of the major hnRNP proteins in the splicing of 56 alternative splicing events in apoptotic genes. Out of this total of 784 alternative splicing reactions tested in three human cell lines, 31 responded similarly to a knockdown in at least two different cell lines. On the other hand, the impact of other hnRNP knockdowns was cell line specific. The broadest effects were obtained with hnRNP K and C, two proteins whose role in alternative splicing had not previously been firmly established. Different hnRNP proteins affected distinct sets of targets with little overlap even between closely related hnRNP proteins. Overall, our study highlights the potential contribution of all of these major hnRNP proteins in alternative splicing control and shows that the targets for individual hnRNP proteins can vary in different cellular contexts.

Introns within Ribosomal Protein Genes Regulate the Production and Function of Yeast Ribosomes

In budding yeast, the most abundantly spliced pre-mRNAs encode ribosomal proteins (RPs). To investigate the contribution of splicing to ribosome production and function, we systematically eliminated introns from all RP genes to evaluate their impact on RNA expression, pre-rRNA processing, cell growth, and response to stress. The majority of introns were required for optimal cell fitness or growth under stress. Most introns are found in duplicated RP genes, and surprisingly, in the majority of cases, deleting the intron from one gene copy affected the expression of the other in a nonreciprocal manner. Consistently, 70% of all duplicated genes were asymmetrically expressed, and both introns and gene deletions displayed copy-specific phenotypic effects. Together, our results indicate that splicing in yeast RP genes mediates intergene regulation and implicate the expression ratio of duplicated RP genes in modulating ribosome function.

Direct detection of alternative open reading frames translation products in human significantly expands the proteome.

A fully mature mRNA is usually associated to a reference open reading frame encoding a single protein. Yet, mature mRNAs contain unconventional alternative open reading frames (AltORFs) located in untranslated regions (UTRs) or overlapping the reference ORFs (RefORFs) in non-canonical +2 and +3 reading frames. Although recent ribosome profiling and footprinting approaches have suggested the significant use of unconventional translation initiation sites in mammals, direct evidence of large-scale alternative protein expression at the proteome level is still lacking. To determine the contribution of alternative proteins to the human proteome, we generated a database of predicted human AltORFs revealing a new proteome mainly composed of small proteins with a median length of 57 amino acids, compared to 344 amino acids for the reference proteome. We experimentally detected a total of 1,259 alternative proteins by mass spectrometry analyses of human cell lines, tissues and fluids. In plasma and serum, alternative proteins represent up to 55% of the proteome and may be a potential unsuspected new source for biomarkers. We observed constitutive co-expression of RefORFs and AltORFs from endogenous genes and from transfected cDNAs, including tumor suppressor p53, and provide evidence that out-of-frame clones representing AltORFs are mistakenly rejected as false positive in cDNAs screening assays. Functional importance of alternative proteins is strongly supported by significant evolutionary conservation in vertebrates, invertebrates, and yeast. Our results imply that coding of multiple proteins in a single gene by the use of AltORFs may be a common feature in eukaryotes, and confirm that translation of unconventional ORFs generates an as yet unexplored proteome.

Identification and comparative analysis of telomerase RNAs from Candida species reveal conservation of functional elements

The RNA component of telomerase (telomerase RNA; TER) varies substantially both in sequence composition and size (from approximately 150 nucleotides [nt] to >1500 nt) across species. This dramatic divergence has hampered the identification of TER genes and a large-scale comparative analysis of TER sequences and structures among distantly related species. To identify by phylogenetic analysis conserved sequences and structural features of TER that are of general importance, it is essential to obtain TER sequences from evolutionarily distant groups of species, providing enough conservation within each group and enough variation among the groups. To this end, we identified TER genes in several yeast species with relatively large (>20 base pairs) and nonvariant telomeric repeats, mostly from the genus Candida. Interestingly, several of the TERs reported here are longer than all other yeast TERs known to date. Within these TERs, we predicted a pseudoknot containing U-A.U base triples (conserved in vertebrates, budding yeasts, and ciliates) and a three-way junction element (conserved in vertebrates and budding yeasts). In addition, we identified a novel conserved sequence (CS2a) predicted to reside within an internal-loop structure, in all the budding yeast TERs examined. CS2a is located near the Est1p-binding bulge-stem previously identified in Saccharomyces cerevisiae. Mutational analyses in both budding yeasts S. cerevisiae and Kluyveromyces lactis demonstrate that CS2a is essential for in vivo telomerase function. The comparative and mutational analyses of conserved TER elements reported here provide novel insights into the structure and function of the telomerase ribonucleoprotein complex.

Alternative splicing of SYK regulates mitosis and cell survival

Most human genes produce multiple mRNA isoforms through alternative splicing. However, the biological relevance of most splice variants remains unclear. In this study, we evaluated the functional impact of alternative splicing in cancer cells. We modulated the splicing pattern of 41 cancer-associated splicing events and scored the effects on cell growth, viability and apoptosis, identifying three isoforms essential for cell survival. Specifically, changing the splicing pattern of the spleen tyrosine kinase gene (SYK) impaired cell-cycle progression and anchorage-independent growth. Notably, exposure of cancer cells to epithelial growth factor modulated the SYK splicing pattern to promote the pro-survival isoform that is associated with cancer tissues in vivo. The data suggest that splicing of selected genes is specifically modified during tumor development to allow the expression of isoforms that promote cancer cell survival.

Retropseudogenes derived from the human Ro/SS-A autoantigen-associated hY RNAs

We report the characterization in the human genome of 966 pseudogenes derived from the four human Y (hY) RNAs, components of the Ro/SS-A autoantigen. About 95% of the Y RNA pseudogenes are found in corresponding locations on the chimpanzee and human chromosomes. On the contrary, Y pseudogenes in mice are both infrequent and found in different genomic regions. In addition to this rodent/primate discrepancy, the conservation of hY pseudogenes relative to hY genes suggests that they occurred after rodent/primate divergence. Flanking regions of hY pseudogenes contain convincing evidence for involvement of the L1 retrotransposition machinery. Although Alu elements are found in close proximity to most hY pseudogenes, these are not chimeric retrogenes. Point mutations in hY RNA transcripts specifically affecting binding of Ro60 protein likely contributed to their selection for direct trans retrotransposition. This represents a novel requirement for the selection of specific RNAs for their genomic integration by the L1 retrotransposition machinery. Over 40% of the hY pseudogenes are found in intronic regions of protein-coding genes. Considering the functions of proteins known to bind subsets of hY RNAs, hY pseudogenes constitute a new class of L1-dependent non-autonomous retroelements, potentially involved in post-transcriptional regulation of gene expression.

HAltORF: a database of predicted out-of-frame alternative open reading frames in human

Human alternative open reading frames (HAltORF) is a publicly available and searchable online database referencing putative products of out-of-frame alternative translation initiation (ATI) in human mRNAs. Out-of-frame ATI is a process by which a single mRNA encodes independent proteins, when distinct initiation codons located in different reading frames are recognized by a ribosome to initiate translation. This mechanism is largely used in viruses to increase the coding potential of small viral genomes. There is increasing evidence that out-of-frame ATI is also used in eukaryotes, including human, and may contribute to the diversity of the human proteome. HAltORF is the first web-based searchable database that allows thorough investigation in the human transcriptome of out-of-frame alternative open reading frames with a start codon located in a strong Kozak context, and are thus the more likely to be expressed. It is also the first large scale study on the human transcriptome to successfully predict the expression of out-of-frame ATI protein products that were previously discovered experimentally. HAltORF will be a useful tool for the identification of human genes with multiple coding sequences, and will help to better define and understand the complexity of the human proteome. Database URL: http://haltorf.roucoulab.com/.

Tumor microenvironment–associated modifications of alternative splicing

Pre-mRNA alternative splicing is modified in cancer, but the origin and specificity of these changes remain unclear. Here, we probed ovarian tumors to identify cancer-associated splicing isoforms and define the mechanism by which splicing is modified in cancer cells. Using high-throughput quantitative PCR, we monitored the expression of splice variants in laser-dissected tissues from ovarian tumors. Surprisingly, changes in alternative splicing were not limited to the tumor tissues but were also found in the tumor microenvironment. Changes in the tumor-associated splicing events were found to be regulated by splicing factors that are differentially expressed in cancer tissues. Overall, ∼20% of the alternative splicing events affected by the down-regulation of the splicing factors QKI and RBFOX2 were altered in the microenvironment of ovarian tumors. Together, our results indicate that the tumor microenvironment undergoes specific changes in alternative splicing orchestrated by a limited number of splicing factors.