Biology subjects

Rodell, R.

Publications and source records attributed to Rodell, R..

5 recordsLinked to original sources

bioRxiv2025

Nano-Mod-Amp reveals RNA sequence, structural and cell type specific features of pseudouridylation by PUS7

Pseudouridines are abundant mRNA modifications that can impact splicing, translation, and stability to tune gene expression. PUS7 is one of the major mRNA pseudouridine synthase whose dysregulation leads to neurodevelopmental disorders and cancer, underscoring the critical function of PUS7-dependent pseudouridines. Beyond a short and degenerate consensus sequence, the molecular mechanisms underlying PUS7-mediated pseudouridylation remain unknown. A lack of targeted, high-throughput pseudouridine detection methods limits simultaneous interrogation of PUS7 regulatory features across many experimental conditions. We developed novel Nanopore sequencing tools, including Nano-Mod-Amp, to reveal pseudouridine stoichiometry, its RNA structural context, and dependence on PUS7 levels at specific sites across biological conditions. We identified a novel RNA structural signature that is associated with more efficient mRNA modification by PUS7. Pseudouridines are largely responsive to modulations in PUS7 protein levels, demonstrating the regulatory potential of varying PUS7 levels across cellular conditions. Conversely, PUS7 activity is also regulated in a cell-type specific manner, independent of PUS7 expression levels in a manner consistent with regulation by RNA structure and RNA binding proteins. Together, we developed Nanopore sequencing tools and uncovered new mechanisms of PUS7 regulation with a framework that can be applied to other RNA-modifying enzymes to query the regulation of the epitranscriptome. HighlightsO_LINanopore direct RNA sequencing identifies PUS7-dependent pseudouridines with stoichiometry. C_LIO_LINano-Mod-Amp quantifies PUS7-dependent pseudouridines at hundreds of sites in high-throughput. C_LIO_LIMPRAs define RNA sequence and structural features associated with modification by PUS7. C_LIO_LIIndividual PUS7 target pseudouridines are substoichiometric and poised for regulation. C_LIO_LIPUS7 activity is regulated by cell type in the absence of differences in PUS7 protein levels. C_LI

molecular biology↗

bioRxiv2025

Nuclear Hormone Receptor NHR-49/HNF4α Couples Fertility Regulation to Resource Allocation and Longevity in C. elegans

The nuclear hormone receptor NHR-49, a homolog of mammalian PPAR and HNF4, is a key transcriptional regulator of nutrition sensing and fatty acid metabolism in Caenorhabditis elegans. Here we uncovered a new function of NHR-49 in reproduction - controlling oocyte activation and ovulation. Loss of NHR-49 causes inappropriate oocyte activation and laying of unfertilized oocytes in the absence of sperm, resulting in rapid loss of yolk and stored fat, and drastically shortening of lifespan. We further demonstrated that prevention of yolk transfer into the oocytes largely restore fat storage and partially rescue lifespan in the nhr-49 mutants. Additionally, NHR-49 appears to couple germline proliferation to nutritional status, as evidenced by its requirement for starvation-induced reduction in germline proliferation. Mechanistically, we showed that NHR-49 primarily acts in somatic cells, rather than the germline itself, to regulate oocyte activation and ovulation. We further demonstrated that NHR-49 binds to the promoter of GSA-1 and may stimulate its expression. GSA-1 encodes a G-protein coupled receptor known to act in the gonadal sheath cells to couple sperm sensing and oocyte activation. Our findings therefore suggest a model whereby NHR-49 regulates the expression of GSA-1, which in turn regulates oocyte activation in response to sperm signal. Overall, our findings suggest a mechanistic link between nutrition sensing and fertility and point to regulated retention of reproductive resources to be critical for maintaining longevity. HighlightsO_LIA new role of NHR-49 in regulating oocyte activation and ovulation C_LIO_LIInappropriate laying of unfertilized oocytes contributes to the fat loss and lifespan shortening of nhr-49 mutants C_LIO_LINHR-49 acts from somatic cells, not germline, to restrain oocyte activation C_LIO_LINHR-49 binds to the promoter of GSA-1 and likely represses gsa-1 expression to regulate oocyte activation C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/664468v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1b965borg.highwire.dtl.DTLVardef@12c61a8org.highwire.dtl.DTLVardef@bf76d7org.highwire.dtl.DTLVardef@133120_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO NHR-49 represses GSA-1 in sheath cells to regulate oocyte activation: We propose that NHR-49 responds to nutritional cues and represses the expression of GSA-1 in sheath cells, which results in aberrant oocyte activation and ovulation. This leads to yolk/fat loss and shortening of parental lifespan. C_FIG

developmental biology↗

bioRxiv2024

YTHDC1 cooperates with the THO complex to prevent RNA damage-induced DNA breaks

Certain environmental toxins are nucleic acid damaging agents, as are many chemotherapeutics used for cancer therapy. These agents induce various adducts in DNA as well as RNA. Indeed, most of the nucleic acid adducts (>90%) formed due to these chemicals, such as alkylating agents, occur in RNA1. However, compared to the well-studied mechanisms for DNA alkylation repair, the biological consequences of RNA damage are largely unexplored. Here, we demonstrate that RNA damage can directly result in loss of genome integrity. Specifically, we show that a human YTH domain-containing protein, YTHDC1, regulates alkylation damage responses in association with the THO complex (THOC)2. In addition to its established binding to N6-methyladenosine (m6A)-containing RNAs, YTHDC1 binds to N1-methyladenosine (m1A)-containing RNAs upon alkylation. In the absence of YTHDC1, alkylation damage results in increased alkylation damage sensitivity and DNA breaks. Such phenotypes are fully attributable to RNA damage, since an RNA-specific dealkylase can rescue these phenotypes. These RNA damage-induced DNA breaks (RDIBs) depend on R-loop formation, which in turn are processed by factors involved in transcription-coupled nucleotide excision repair. Strikingly, in the absence of YTHDC1 or THOC, an RNA m1A methyltransferase targeted to the nucleus is sufficient to induce DNA breaks. Our results uncover a unique role for YTHDC1-THOC in base damage responses by preventing RDIBs, providing definitive evidence for how damaged RNAs can impact genomic integrity.

molecular biology↗

bioRxiv2021

The ASCC2 CUE domain contacts adjacent ubiquitins to recognize K63-linked polyubiquitin

Alkylation of DNA and RNA is a potentially toxic lesion that can result in mutations and cell death. In response to alkylation damage, K63-linked polyubiquitin chains are assembled that localize the ALKBH3-ASCC repair complex to damage sites in the nucleus. The protein ASCC2, a subunit of the ASCC complex, selectively binds K63-linked polyubiquitin chains using its CUE domain, a type of ubiquitin-binding domain that typically binds monoubiquitin and does not discriminate among different polyubiquitin linkage types. We report here that the ASCC2 CUE domain selectively binds K63-linked diubiquitin by contacting both the distal and proximal ubiquitin. Whereas the ASCC2 CUE domain binds the distal ubiquitin in a manner similar to that reported for other CUE domains bound to a single ubiquitin, the contacts with the proximal ubiquitin are unique to ASCC2. The N-terminal portion of the ASCC2 1 helix, including residues E467 and S470, contributes to the binding interaction with the proximal ubiquitin of K63-linked diubiquitin. Mutation of residues within the N-terminal portion of the ASCC2 1 helix decreases ASCC2 recruitment in response to DNA alkylation, supporting the functional significance of these interactions during the alkylation damage response.

biophysics↗

bioRxiv2020

Aberrant RNA methylation triggers recruitment of an alkylation repair complex

A critical question in genome stability is the nature of the chemical damage responsible for repair activation. We previously reported a novel pathway specifically activated during alkylation damage in human cells, where the E3 ubiquitin ligase RNF113A mediates the recruitment of the ASCC repair complex. Yet the mechanistic basis for the alkylation damage selectivity of this pathway remains unclear. Here, we demonstrate that RNA but not DNA alkylation is the initiating signal for this process. Aberrantly methylated RNA is sufficient to recruit ASCC, while an RNA dealkylase suppresses ASCC recruitment during chemical alkylation. This aberrant RNA methylation causes transcriptional repression in a manner dependent on the ASCC complex. We show that an alkylated pre-mRNA, or an RNA containing a single damaged base, is sufficient to activate RNF113A E3 activity in a phosphorylation-dependent manner. Together, our work identifies an unexpected role for RNA damage in eliciting a DNA repair response, and suggests that RNA may serve as the "canary in the coal mine" for sensing alkylation damage.

molecular biology↗