A organização 4D do splicing do pre-mRNA

The aim of this project is to investigate the spatial and temporal dynamics of pre-mRNAs within the nuclear landscape and translate this knowledge into more efficient splicing modulatory strategies with potential therapeutic applications. The project builds on our recent observation that a significant fraction of nascent transcripts remains unspliced (Fig 1, PMID: 33735606). We will addresses three core questions: 1) What happens to nascent pre-mRNAs that are not rapidly spliced and cleaved? 2) Why are some pre-mRNAs rapidly spliced while others remain unspliced? 3) Can delayed splicing be targeted to modulate splicing outcomes? Specifically, we will: 1.1 Develop an innovative experimental design to image spliced and unspliced transcripts from selected long genes using single-molecule RNA FISH and super-resolution microscopy (Fig 2). 1.2 Quantify the ratio of spliced nascent transcripts to unspliced and uncleaved ones. We will purify nascent transcripts by Pol II immunoprecipitation and design ddPCR assays targeting multiple splice-junctions and past-polyA sequences (Fig 2). 1.3 Induce rapid depletion of essential exosome components and analyse the localization and relative abundance of unspliced pre-mRNAs. If these molecules fail to undergo splicing and export, they should become stabilized in the absence of exosome activity. 2.1 Optimize the use of pharmacological inhibitors of transcription and splicing to enrich for unspliced transcripts in the nucleus. 2.2 Identify RNAs and proteins associated with Pol II under distinct transcription and splicing scenarios. We will use Pol II immunopurification and APEX2 proximity labelling combined with RNA sequencing (short and long reads) and mass spectrometry (Fig 3). 2.3 Visualize the higher-order organization of Pol II, spliceosome components and splicing intermediates by super-resolution microscopy. As a model system, we will use the giant titin gene that is highly expressed in the nucleus of cardiac muscle cells, where it forms a transcription loop with several microns in length making it possible to resolve polymerases moving along the gene axis and carrying nascent RNAs (Fig 4). 3.1 Investigate whether there is a delay between the pioneer rounds of transcription and the onset of splicing. We will use FISH and nascent RNA sequencing (short and long reads) to visualize spliced and unspliced nascent transcripts during the first rounds of transcription immediately after mitosis or after release of drug-induced transcription inhibition. 3.2 Determine the consequences of depleting nascent uncleaved/unspliced transcripts. We will induce the selective degradation of titin uncleaved pre-mRNAs with gapmer antisense oligonucleotides. We will then quantify the proportion of spliced and unspliced nascent titin transcripts and measure abundance of titin-derived non-canonical splice products, including circular RNAs. 3.3 Explore strategies to improve the efficacy of splicing modulatory antisense oligonucleotides (ASOs) by forcing their concentration in the vicinity of the target nascent transcript. As a model system, we will focus on alternative splicing of titin transcripts in cardiac muscle cells. We will design a new generation of ASOs that contain sequences complementary to the target splice site to be modulated and additional sequences that will attract the oligo to the titin transcripts that remain unspliced (Fig 5).