Biology subjects

Smolka, J. A.

Publications and source records attributed to Smolka, J. A..

3 recordsLinked to original sources

bioRxiv2025

Integrating process, proximity, and prediction implicates novel protein and RNA interactions in human Origin Recognition Complex function

Genome replication start sites, called origins, begin to be specified by Origin Recognition Complex (ORC) proteins prior to replication through a process called origin licensing. Once licensed, origins become active and initiate DNA synthesis with varying efficiencies influenced by local chromatin environment, transcription, and 3D genome organization. ORC proteins have also been implicated in regulating chromatin state and nuclear organization. However, it is unclear if there is interplay between ORC and the chromatin architectures underlying origin activation, as we lack a systems-level understanding of how ORC proteins interact, post-licensing, with the nuclear environments conducive to genome synthesis. To infer this context-specific ORC interactome, I used data from genome-wide CRISPR fitness screens, a cell-wide proximity labeling study, and proteomic profiling of nascent DNA-associated proteins to identify 17 novel factors that genetically and proteomically interact with ORC subunits and genome replication. Unexpectedly, the candidate pool was significantly enriched for factors involved in the homeostasis of RNA Polymerase III (Pol III) transcripts, particularly 5S ribosomal RNA (rRNA) and transfer RNA (tRNA). Follow-up protein-protein structure predictions by AlphaFold 3 (AF3) proposed direct interactions between ORC subunits and Pol III transcript biogenesis factors, as well as epigenetic regulators and a cyclin-dependent kinase. Given the prominence of 5S rRNA and tRNA biogenesis factors in my results, and prior reports of ORC subunits binding RNA, I also used protein-RNA structure prediction to identify candidate ORC3 interactions with 5S rRNA and tRNA. Altogether, my analysis integrates biological process, molecular proximity in human cells, and structural prediction to nominate novel protein and RNA interactions for involvement in the human replication program. These results augment and expand current models for ORC function and origin activation, particularly those involving chromatin state and transcriptional activity, and generate testable hypotheses to explore the interdependencies of replication patterning, histone modification, and nuclear RNA homeostasis.

bioinformatics↗

bioRxiv2024

A spatial atlas of mitochondrial gene expression reveals dynamic translation hubs and remodeling in stress

Mitochondrial genome expression is important for cellular bioenergetics. How mitochondrial RNA processing and translation are spatially organized across dynamic mitochondrial networks is not well understood. Here, we report that processed mitochondrial RNAs are consolidated with mitoribosome components into translation hubs distal to either nucleoids or processing granules in human cells. During stress, these hubs are remodeled into translationally repressed mesoscale bodies containing messenger, ribosomal, and double-stranded RNA. We show that the highly conserved helicase SUV3 contributes to the distribution of processed RNA within mitochondrial networks, and that stress bodies form downstream of proteostatic stress in cells lacking SUV3 unwinding activity. We propose that the spatial organization of nascent chain synthesis into discrete domains serves to throttle the flow of genetic information in stress to ensure mitochondrial quality control.

cell biology↗

bioRxiv2020

Recognition of cellular RNAs by the S9.6 antibody creates pervasive artefacts when imaging RNA:DNA hybrids

The contribution of RNA:DNA hybrid metabolism to cellular processes and disease states has become a prominent topic of study. The S9.6 antibody recognizes RNA:DNA hybrids with a subnanomolar affinity, making it a broadly used tool to detect and study RNA:DNA hybrids. However, S9.6 also binds double-stranded RNA in vitro with significant affinity. Though frequently used in immunofluorescence microscopy, the possible reactivity of S9.6 with non-RNA:DNA hybrid substrates in situ, particularly RNA, has not been comprehensively addressed. Furthermore, S9.6 immunofluorescence microscopy has been methodologically variable and generated discordant imaging datasets. In this study, we find that the majority of the S9.6 immunofluorescence signal observed in fixed human cells arises from RNA, not RNA:DNA hybrids. S9.6 staining was quantitatively unchanged by pre-treatment with the human RNA:DNA hybrid-specific nuclease, RNase H1, despite experimental verification in situ that S9.6 could recognize RNA:DNA hybrids and that RNase H1 was active. S9.6 staining was, however, significantly sensitive to pre-treatments with RNase T1, and in some cases RNase III, two ribonucleases that specifically degrade single-stranded and double-stranded RNA, respectively. In contrast, genome-wide maps obtained by high-throughput DNA sequencing after S9.6-mediated DNA:RNA Immunoprecipitation (DRIP) are RNase H1-sensitive and RNase T1- and RNase III-insensitive. Altogether, these data demonstrate that the S9.6 antibody, though capable of recognizing RNA:DNA hybrids in situ and in vitro, suffers from a lack of specificity that precludes reliable imaging of RNA:DNA hybrids and renders associated imaging data inconclusive in the absence of controls for its promiscuous recognition of cellular RNAs.

molecular biology↗