Claudia Ribeiro de Almeida

Biosynthesis of histone messenger RNA employs a specific 3' end endonuclease.
Pettinati I, Grzechnik P, Ribeiro de Almeida C, Brem J, McDonough MA, Dhir S, Proudfoot NJ, Schofield CJ

Replication-dependent (RD) core histone mRNA produced during S-phase is the only known metazoan protein-coding mRNA presenting a 3' stem-loop instead of the otherwise universal polyA tail. A metallo β-lactamase (MBL) fold enzyme, cleavage and polyadenylation specificity factor 73 (CPSF73), is proposed to be the sole endonuclease responsible for 3' end processing of both mRNA classes. We report cellular, genetic, biochemical, substrate selectivity, and crystallographic studies providing evidence that an additional endoribonuclease, MBL domain containing protein 1 (MBLAC1), is selective for 3' processing of RD histone pre-mRNA during the S-phase of the cell cycle. Depletion of MBLAC1 in cells significantly affects cell cycle progression thus identifying MBLAC1 as a new type of S-phase-specific cancer target.

eLife, 7, 2050-084X, 2018

Deregulated Expression of Mammalian lncRNA through Loss of SPT6 Induces R-Loop Formation, Replication Stress, and Cellular Senescence.
Nojima T, Tellier M, Foxwell J, Ribeiro de Almeida C, Tan-Wong SM, Dhir S, Dujardin G, Dhir A, Murphy S, Proudfoot NJ

Extensive tracts of the mammalian genome that lack protein-coding function are still transcribed into long noncoding RNA. While these lncRNAs are generally short lived, length restricted, and non-polyadenylated, how their expression is distinguished from protein-coding genes remains enigmatic. Surprisingly, depletion of the ubiquitous Pol-II-associated transcription elongation factor SPT6 promotes a redistribution of H3K36me3 histone marks from active protein coding to lncRNA genes, which correlates with increased lncRNA transcription. SPT6 knockdown also impairs the recruitment of the Integrator complex to chromatin, which results in a transcriptional termination defect for lncRNA genes. This leads to the formation of extended, polyadenylated lncRNAs that are both chromatin restricted and form increased levels of RNA:DNA hybrid (R-loops) that are associated with DNA damage. Additionally, these deregulated lncRNAs overlap with DNA replication origins leading to localized DNA replication stress and a cellular senescence phenotype. Overall, our results underline the importance of restricting lncRNA expression.

Molecular cell, 72, 1097-4164, 2018

RNA Helicase DDX1 Converts RNA G-Quadruplex Structures into R-Loops to Promote IgH Class Switch Recombination.
Ribeiro de Almeida C, Dhir S, Dhir A, Moghaddam AE, Sattentau Q, Meinhart A, Proudfoot NJ

Class switch recombination (CSR) at the immunoglobulin heavy-chain (IgH) locus is associated with the formation of R-loop structures over switch (S) regions. While these often occur co-transcriptionally between nascent RNA and template DNA, we now show that they also form as part of a post-transcriptional mechanism targeting AID to IgH S-regions. This depends on the RNA helicase DDX1 that is also required for CSR in vivo. DDX1 binds to G-quadruplex (G4) structures present in intronic switch transcripts and converts them into S-region R-loops. This in turn targets the cytidine deaminase enzyme AID to S-regions so promoting CSR. Notably R-loop levels over S-regions are diminished by chemical stabilization of G4 RNA or by the expression of a DDX1 ATPase-deficient mutant that acts as a dominant-negative protein to reduce CSR efficiency. In effect, we provide evidence for how S-region transcripts interconvert between G4 and R-loop structures to promote CSR in the IgH locus.

Molecular cell, 70, 1097-4164, 2018