SMC complexes are widely conserved ATP-powered DNA-loop-extrusion motors indispensable for organizing and faithfully segregating chromosomes. How SMC complexes translocate along DNA for loop extrusion and what happens when two complexes meet on the same DNA molecule is largely unknown. Revealing the origins and the consequences of SMC encounters is crucial for understanding the folding process not only of bacterial, but also of eukaryotic chromosomes. Here, we uncover several factors that influence bacterial chromosome organization by modulating the probability of such clashes. These factors include the number, the strength, and the distribution of Smc loading sites, the residency time on the chromosome, the translocation rate, and the cellular abundance of Smc complexes. By studying various mutants, we show that these parameters are fine-tuned to reduce the frequency of encounters between Smc complexes, presumably as a risk mitigation strategy. Mild perturbations hamper chromosome organization by causing Smc collisions, implying that the cellular capacity to resolve them is limited. Altogether, we identify mechanisms that help to avoid Smc collisions and their resolution by Smc traversal or other potentially risky molecular transactions.
All deep sequencing data has been deposited to the NCBI GEO database and will be available at GEO Accession number: GSE163573All other raw data will be made available via Mendeley Data DOI:10.17632/kvjd6nj2bh.2
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
© 2021, Anchimiuk et al.
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RNA interference (RNAi) is a conserved pathway that utilizes Argonaute proteins and their associated small RNAs to exert gene regulatory function on complementary transcripts. While the majority of germline-expressed RNAi proteins reside in perinuclear germ granules, it is unknown whether and how RNAi pathways are spatially organized in other cell types. Here, we find that the small RNA biogenesis machinery is spatially and temporally organized during Caenorhabditis elegans embryogenesis. Specifically, the RNAi factor, SIMR-1, forms visible concentrates during mid-embryogenesis that contain an RNA-dependent RNA polymerase, a poly-UG polymerase, and the unloaded nuclear Argonaute protein, NRDE-3. Curiously, coincident with the appearance of the SIMR granules, the small RNAs bound to NRDE-3 switch from predominantly CSR-class 22G-RNAs to ERGO-dependent 22G-RNAs. NRDE-3 binds ERGO-dependent 22G-RNAs in the somatic cells of larvae and adults to silence ERGO-target genes; here we further demonstrate that NRDE-3-bound, CSR-class 22G-RNAs repress transcription in oocytes. Thus, our study defines two separable roles for NRDE-3, targeting germline-expressed genes during oogenesis to promote global transcriptional repression, and switching during embryogenesis to repress recently duplicated genes and retrotransposons in somatic cells, highlighting the plasticity of Argonaute proteins and the need for more precise temporal characterization of Argonaute-small RNA interactions.
A new method for mapping torsion provides insights into the ways that the genome responds to the torsion generated by RNA polymerase II.