NRDE2 negatively regulates exosome functions by inhibiting MTR4 recruitment and exosome interaction.

The exosome functions in the degradation of diverse RNA species, yet how it is negatively regulated remains largely unknown. Here, we show that NRDE2 forms a 1:1 complex with MTR4, a nuclear exosome cofactor critical for exosome recruitment, via a conserved MTR4-interacting domain (MID). Unexpectedly, NRDE2 mainly localizes in nuclear speckles, where it inhibits MTR4 recruitment and RNA degradation, and thereby ensures efficient mRNA nuclear export. Structural and biochemical data revealed that NRDE2 interacts with MTR4's key residues, locks MTR4 in a closed conformation, and inhibits MTR4 interaction with the exosome as well as proteins important for MTR4 recruitment, such as the cap-binding complex (CBC) and ZFC3H1. Functionally, MID deletion results in the loss of self-renewal of mouse embryonic stem cells. Together, our data pinpoint NRDE2 as a nuclear exosome negative regulator that ensures mRNA stability and nuclear export.

Mouse nuclear RNAi-defective 2 promotes splicing of weak 5' splice sites.

Removal of introns during pre-mRNA splicing, which is central to gene expression, initiates by base pairing of U1 snRNA with a 5' splice site (5'SS). In mammals, many introns contain weak 5'SSs that are not efficiently recognized by the canonical U1 snRNP, suggesting alternative mechanisms exist. Here, we develop a cross-linking immunoprecipitation coupled to a high-throughput sequencing method, BCLIP-seq, to identify NRDE2 (nuclear RNAi-defective 2), and CCDC174 (coiled-coil domain-containing 174) as novel RNA-binding proteins in mouse ES cells that associate with U1 snRNA and 5'SSs. Both proteins bind directly to U1 snRNA independently of canonical U1 snRNP-specific proteins, and they are required for the selection and effective processing of weak 5'SSs. Our results reveal that mammalian cells use noncanonical splicing factors bound directly to U1 snRNA to effectively select suboptimal 5'SS sequences in hundreds of genes, promoting proper splice site choice, and accurate pre-mRNA splicing.

NRDE-2, the human homolog of fission yeast Nrl1, prevents DNA damage accumulation in human cells.

The RNA helicase Mtr4 is a versatile protein that is a crucial component of several distinct RNA surveillance complexes. Here we describe a novel complex that contains Mtr4, but has a role distinct from any of those previously described. We found that Mtr4 association with the human homolog of fission yeast Nrl1, NRDE-2, defines a novel function for Mtr4 in the DNA damage response pathway. We provide biochemical evidence that Mtr4 and NRDE-2 are part of the same complex and show that both proteins play a role in the DNA damage response by maintaining low DNA double-strand break levels. Importantly, the DNA damage response function of the Mtr4/NRDE-2 complex does not depend on the formation of R loops. We show however that NRDE-2 and Mtr4 can affect R-loop signals at a subset of distinct genes, possibly regulating their expression. Our work not only expands the wide range of Mtr4 functions, but also elucidates an important role of the less characterized human NRDE-2 protein.

NRDE2 deficiency impairs homologous recombination repair and sensitizes hepatocellular carcinoma to PARP inhibitors.

To identify novel susceptibility genes for hepatocellular carcinoma (HCC), we performed a rare-variant association study in Chinese populations consisting of 2,750 cases and 4,153 controls. We identified four HCC-associated genes, including NRDE2, RANBP17, RTEL1, and STEAP3. Using NRDE2 (index rs199890497 [p.N377I], p = 1.19 × 10-9) as an exemplary candidate, we demonstrated that it promotes homologous recombination (HR) repair and suppresses HCC. Mechanistically, NRDE2 binds to the subunits of casein kinase 2 (CK2) and facilitates the assembly and activity of the CK2 holoenzyme. This NRDE2-mediated enhancement of CK2 activity increases the phosphorylation of MDC1 and then facilitates the HR repair. These functions are eliminated almost completely by the NRDE2-p.N377I variant, which sensitizes the HCC cells to poly(ADP-ribose) polymerase (PARP) inhibitors, especially when combined with chemotherapy. Collectively, our findings highlight the relevance of the rare variants to genetic susceptibility to HCC, which would be helpful for the precise treatment of this malignancy.

A Conserved NRDE-2/MTR-4 Complex Mediates Nuclear RNAi in Caenorhabditis elegans.

Small regulatory RNAs, such as small interfering RNAs (siRNAs) and PIWI-interacting RNAs, regulate splicing, transcription, and genome integrity in many eukaryotes. In Caenorhabditis elegans, siRNAs bind nuclear Argonautes (AGOs), which interact with homologous premessenger RNAs to recruit downstream silencing effectors, such as NRDE-2, to direct cotranscriptional gene silencing [or nuclear RNA interference (RNAi)]. To further our understanding of the mechanism of nuclear RNAi, we conducted immunoprecipitation-mass spectrometry on C. elegans NRDE-2 The major NRDE-2 interacting protein identified was the RNA helicase MTR-4 Co-immunoprecipitation analyses confirmed a physical association between NRDE-2 and MTR-4 MTR-4 colocalizes with NRDE-2 within the nuclei of most/all C. elegans somatic and germline cells. MTR-4 is required for nuclear RNAi, and interestingly, MTR-4 is recruited to premessenger RNAs undergoing nuclear RNAi via a process requiring nuclear siRNAs, the nuclear AGO HRDE-1, and NRDE-2, indicating that MTR-4 is a component of the C. elegans nuclear RNAi machinery. Finally, we confirm previous reports showing that human (Hs)NRDE2 and HsMTR4 also physically interact. Our data show that the NRDE-2/MTR-4 interactions are evolutionarily conserved, and that, in C. elegans, the NRDE-2/MTR-4 complex contributes to siRNA-directed cotranscriptional gene silencing.

Identification of proteins associated with splicing factors Ntr1, Ntr2, Brr2 and Gpl1 in the fission yeast Schizosaccharomyces pombe.

The spliceosome is a complex molecular machine assembled from many components, which catalyzes the removal of introns from mRNA precursors. Our previous study revealed that the Nrl1 (NRDE-2 like 1) protein associates with spliceosome proteins and regulates pre-mRNA splicing and homologous recombination-dependent R-loop formation in the fission yeast Schizosaccharomyces pombe. Here, we identify proteins associated with splicing factors Ntr1, Ntr2, Brr2 and Gpl1, a poorly characterized G-patch domain-containing protein required for efficient splicing. This work provides new evidence that Nrl1 and splicing factors physically interact and reveals additional insights into the protein interaction network of the spliceosome. We discuss implications of these findings in the light of recent progress in our understanding of how Nrl1 and splicing factors ensure genome stability.