Talks by Eric ALLEMAND and Martin DUTERTRE

Eric ALLEMAND
“Laboratory of cellular and molecular mechanisms of hematological disorders and therapeutic implications, U1163-CEREMAST, Foundation IMAGINE”

Title:
Regulation of transcriptome diversity through alternative splicing and its role in hematological genetic disorders.

Abstract:
RNA processing, particularly alternative splicing, regulates nearly all human genes and is a key contributor to gene expression diversity. Splicing defects are implicated in around 35% of genetic diseases and play an important role in cellular transformation, especially in cancers arising from impaired hematopoietic differentiation. In myelodysplastic syndromes and mastocytosis, pathogenic mutations frequently target splicing factors, while widespread splicing deregulation is observed in leukemia and lymphoma. Although high-throughput Exome and Whole Genome Sequencing (WGS) with short-read technologies have revolutionized mutation detection, these methods are inherently limited in their capacity to thoroughly analyze splicing defects. We are developing projects that aim to unravel splicing dysregulation in hematological disorders using Third Generation Sequencing (TGS) technologies. Our focus is particularly directed toward transcripts whose expression constitutes a minor fraction of the full repertoire of splice variants. These approaches reveal an underappreciated diversity in the transcriptome that is often missed by conventional genetic analyses. Our work seeks to identify novel pathological markers linked to splicing regulation, explore their functional impacts, and develop corrective tools to address these defects. We work on: Identification of aberrant splice-driven signature in T-cell acute lymphoblastic leukemia. Reprogramming the post-transcriptional regulation of BCL11A to treat β-hemoglobinopathies. Global analysis of alternative splicing in erythropoiesis: unveiling novel therapeutic approaches to β-hemoglobinopathies.

Martin DUTERTRE
“RNA Biology, Signaling and Cancer” Lab, INSERM U1278, UMR 3348 CNRS, Institut Curie - Centre de Recherche, ORSAY, FRANCE, martin.dutertre@curie.fr

Title: Microprotein-coding intronic polyadenylation isoforms: A new genetic paradigm

Abstract:
On the one hand, many human genes generate intronic polyadenylation (IPA) isoforms, that terminate in an alternative last exon and often encode isoforms of canonical proteins. On the other hand, microproteins are an emerging class of small proteins (about 100 amino acids or less) that are translated from small open reading frames (sORFs) located in annotated noncoding RNAs and canonical mRNAs. However, microprotein production by IPA transcripts has not been studied. In this study, based on multiple omics analyses (3’-seq on total RNA and polysome fractions, short- and long-read RNA-seq, Ribo-seq and mass spectrometry), we reveal that IPA isoforms are a novel source of microproteins. Indeed, we show that many human protein-coding genes generate a microprotein-coding IPA transcript (coined miP-5’UTR-IPA isoform), from the same promoter as the canonical mRNA but using a polyadenylation site located in an intron within the annotated 5’-untranslated region of the gene. By 3’-seq on lung cancer cells, we show that cisplatin (a genotoxic agent commonly used to treat this cancer) favors the expression of many miP-5’UTR-IPA isoforms relative to matched canonical mRNAs, through an inhibition of transcription processivity in a FANCD2 and senataxin-dependent manner. We further characterized the miP-5’UTR-IPA isoform of one of these genes, PRKAR1B: we detected the encoded microprotein; CRISPR editing of either the IPA site or the sORF initiation codon in A549 cells led to a decrease of cell growth inhibition by cisplatin; motif analysis in the microprotein predicted its interaction with two p53-regulatory proteins; and accordingly, CRISPR clones showed decreased p53 protein induction by cisplatin. Finally, we built a database of human miP-5’UTR-IPA isoforms and show their expression in normal tissues and cell types. Altogether, these findings reveal the novel paradigm of miP-5’UTR-IPA genes, provide the proof of principle of their functionality and suggest their role in cancer cell response to a genotoxic agent.