Development of novel approaches for the study of RNA dynamics
The characterization of RNA dynamics is typically based on the joint profiling of total and nascent RNAs, the latter profiled through RNA metabolic labeling. The integrative analysis of these data, which ultimately allows disentangling pre-existing from newly synthetized transcripts, allows us to decipher how gene expression programs are established by the balance of synthesis, processing and degradation. Achievements and ongoing activity:
- The development of INSPEcT (de Pretis S et al., Bioinformatics 2015), which allowed for the first time the comprehensive analysis of RNA dynamics.
- We developed a novel version of INSPEcT, which allows the quantification of RNA dynamics without requiring the quantification of nascent RNA (Furlan M et al., Genome Res 2020).
- INSPEcT was used to characterize how the activation of MYC impacts RNA dynamics (de Pretis et al., Genome Res 2017; Tesi A et al., EMBO Reports 2019). These studies provided unprecedented details on how this fundamental transcription factor and oncogene controls the transcription of thousands of target genes.
Development of novel approaches for the study of RNA Polymerase II (RNAPII) dynamics
RNAPII is a key actor in genes’ transcription and a complex regulatory hub. The life-cycle of the RNAPII complex includes its recruitment and assembly to promoters, followed by pause-release, elongation, and detachment from the 3’ end of genes. Most of the studies dealing with the RNAPII life-cycle rely on ChIP-seq data, while it has been shown that these are unable to univocally quantify the dynamics of each step. For example, a variation in the density of RNAPII bound to promoters can be due to a change in either its recruitment or in its pause-release rate (or both). We recently developed a method to properly address this issue (de Pretis et al., Genome Res 2017), which was used to provide critical insights on the mechanisms through which MYC modulates its targets. Achievements and ongoing activity:
- The development of a computational method that allows the quantification of the kinetic rates of RNAPII recruitment, pause-release, elongation and detachment (de Pretis et al., Genome Res 2017).
- This method, applied in the context of MYC activation, revealed that the most prominent effect of the binding of this factor is not the promotion of pause-release, as previously suggested, while RNAPII recruitment (de Pretis et al., Genome Res 2017; Tesi A. et al., EMBO Reports 2019). Moreover, this suggested that MYC primarily acts as an activator, as recently independently confirmed. These data indeed suggest that the repression of a subset of MYC target genes is mostly a passive consequence acute MYC activation.
- We are developing a novel method, relying on RNA metabolic labeling and single molecule Nanopore sequencing, to study the impact of RNA modifications on RNA and RNAPII dynamics at the level of single isoforms.
The role of the m6A-epitranscriptome in cancer
Despite the rapid progress in the field, various aspects of the functional role of this mark remain poorly characterized. We are far from a complete understanding of how m6A influences the dynamics of marked transcripts, and we are just at the beginning in understanding the crosstalk between the m6A machinery and chromatin regulation. Moreover, and more importantly, we just started appreciating how the epitranscriptome could shape aberrant gene expression programs in cancer. Ongoing activity in the lab, supported through a grant and a fellowship from AIRC:
- We are studying the role of the m6A epitranscriptome in the context of MYC-driven aberrant transcriptional programs in breast cancer.
- We are characterizing the role of the m6A epitranscriptome in the context of drug resistance in Hepatocellular Carcinoma.