Biology subjects

Alagia, A.

Publications and source records attributed to Alagia, A..

5 recordsLinked to original sources

bioRxiv2026

Structural and functional basis of the non-canonical human Dicer-tRNA complex

Human Dicer (hDicer) is a key enzyme in the RNA interference (RNAi) pathway that generates [~]21-22 nt micro-RNA (miRNAs) and small interfering RNAs (siRNAs). We have previously shown that hDicer also generates tRNA-derived small RNAs (tsRNAs), which mediate nuclear gene silencing and regulate hundreds of disease-associated genes. As powerful and evolutionarily conserved cellular regulators, tsRNAs emerged as an important class of small RNAs. Therefore, it is essential to understand their biogenesis. However, the molecular and structural basis of tRNA cleavage by hDicer, as well as the role of chemical modifications such as 5-methylcytosine (m5C), in this process, remain unknown. Here, we present the first structural insights into hDicer in complex with tRNA, obtained by cryo-electron microscopy (cryo-EM), selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) and molecular dynamics (MD) simulations. Our results reveal that tRNAs adopt alternative conformations that are recognized and processed by hDicer. Furthermore, we show that tRNA cleavage by hDicer is facilitated by the m5C modification deposited by Nop2/SUN RNA methyltransferase 2 (NSUN2). Collectively, our findings redefine tRNAs as bona fide hDicer substrates and uncover a modification-dependent biogenetic pathway that reshapes the current understanding of the origins and regulation of human small RNAs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=73 SRC="FIGDIR/small/744379v1_ufig1.gif" ALT="Figure 1000"> View larger version (24K): org.highwire.dtl.DTLVardef@14a8682org.highwire.dtl.DTLVardef@1c4d0daorg.highwire.dtl.DTLVardef@6bc433org.highwire.dtl.DTLVardef@11e27e1_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

NSUN2 Facilitates DICER Cleavage of DNA Damage-Associated R-Loops to Promote Repair

DNA integrity is constantly challenged by both endogenous and exogenous damaging agents, resulting in various forms of damage. Failure to repair DNA accurately leads to genomic instability, a hallmark of cancer. Distinct pathways exist to repair different types of DNA damage. Double-strand breaks (DSBs) represent particularly severe form of damage, due to the physical separation of DNA strands. The repair of DSBs requires the activity of RNA Polymerase II (RNAPII) and the generation of Damage-associated transcripts (DARTs). Here we show that the RNA m5C-methyltransferase NSUN2 localizes to DSBs in a transcription-dependent manner, where it binds to and methylates DARTs. The depletion of NSUN2 results in an accumulation of nascent primary DARTs around DSBs. Furthermore, we detected an RNA-dependent interaction between NSUN2 and DICER, which was stimulated by DNA damage. NSUN2 activity promoted DICER cleavage of DARTs-associated R-loops, which is required for efficient DNA repair. We report a previously unrecognized role of the RNA m5C-methyltransferase NSUN2 within the RNA-dependent DNA damage response, highlighting its function as a DICER chaperone for the clearance of non-canonical substrates such as DARTs, thereby contributing to genomic integrity.

molecular biology↗

bioRxiv2024

TIRR regulates mRNA export and association with P bodies in response to DNA damage

To ensure the integrity of our genetic code, a coordinated network of signalling and repair proteins known as the DNA damage response (DDR) detects and repairs DNA insults, the most toxic being double-stranded breaks (DSBs). Tudor interacting repair regulator (TIRR) is a key factor in DSB repair, acting through its interaction with p53 binding protein 1 (53BP1). TIRR is also an RNA-binding protein, yet its role in RNA regulation during the DNA damage response remains elusive. Here we show that TIRR selectively binds to a subset of mRNAs in response to DNA damage with preference for transcripts encoding transcription factors and RNA polymerase II (RNAPII) transcription regulators. Upon DNA damage, TIRR interacts with the nuclear export protein Exportin-1 (XPO1), through its nuclear export sequence (NES). Furthermore, TIRR plays a crucial role in modulation of RNA processing bodies (P bodies/PBs). TIRR itself and TIRR-bound RNA co-localises with PBs, and TIRR depletion results in nuclear RNA retention and impaired PB formation. Finally, the role of TIRR in RNA export contributes to efficient DNA damage response. This work reveals intricate involvement of TIRR in orchestrating mRNA nuclear export and storage within PBs, emphasizing its significance in the regulation of RNA-mediated DNA damage response.

molecular biology↗

bioRxiv2023

Phosphorylated trimeric SOSS1 complex and RNA polymerase II trigger liquid-liquid phase separation at double-strand breaks

The most toxic forms of DNA damage are double-strand breaks (DSBs). We have previously shown that RNA polymerase II (RNAPII), phosphorylated at tyrosine 1 (Y1P) on the C- terminal domain, transcribes RNA at DSBs to promote efficient DNA repair. However, it is still unknown how transcription is regulated at DSBs. Here, we show that the trimeric SOSS1 complex (hSSB1, INTS3, and c9orf80) binds to Y1P RNAPII in response to DNA damage, hSSB1 binds to R-loops, and formation of the SOSS1 complex is required for the coexistence of replication protein A (RPA) and hSSB1 at DSBs. The damage-activated tyrosine kinase c- Abl phosphorylates hSSB1 to enable its binding to Y1P RNAPII and its recruitment to DSBs. Finally, we show both in vitro and in vivo that the SOSS1 complex and RNAPII form dynamic repair compartments at DSBs via liquid-liquid phase separation (LLPS). The loss of the trimeric SOSS1 leads to impaired DNA repair, highlighting its biological importance in the RNA-dependent DNA damage response. TeaserTrimeric SOSS1 complex and transcription contribute to phase separation at double-strand DNA breaks.

molecular biology↗

bioRxiv2022

Small non-coding vault RNA1-2 modulate expression of cell membrane proteins through nascent RNA silencing

Gene expression can be regulated by transcriptional or post-transcriptional gene silencing. Recently, we described nuclear nascent RNA silencing (NRS) that is mediated by Dicer dependent tRNA-derived small RNA molecules. In addition to tRNA, RNA polymerase III also transcribes Vault RNA, a component of the ribonucleoprotein complex Vault. Here, we show that Dicer dependent small vault RNA1-2 (svtRNA1-2) associate with Argonaute 2 (Ago2). Whilst endogenous vtRNA1-2 is present mostly in cytoplasm, svtRNA1-2 localises predominantly in nucleus. Furthermore, in Ago2 and Dicer knockdown cells, a subset of genes which are upregulated at the nascent level were predicted to be targeted by svtRNA1-2 in the intronic region. Genomic deletion of vtRNA1-2 results in impaired cellular proliferation and the upregulation of genes associated with cell membrane physiology and cell adhesion. Silencing activity of svtRNA1-2 molecules is dependent on seed-plus-complementary-paired hybridisation features and the presence of a 5-nucleotide loop protrusion on target RNAs. Our data reveal a role for Dicer dependent svtRNA1-2, possessing unique molecular features, in modulation of expression of membrane associated proteins at the nascent RNA level.

molecular biology↗