Biology subjects

Kirstein, N.

Publications and source records attributed to Kirstein, N..

3 recordsLinked to original sources

bioRxiv2021

The Integrator complex regulates microRNA abundance through RISC loading

MicroRNA (miRNA) homeostasis is crucial for the post-transcriptional regulation of their target genes and miRNA dysregulation has been linked to multiple diseases, including cancer. The molecular mechanisms underlying miRNA biogenesis from processing of primary miRNA transcripts to formation of mature miRNA duplex are well understood1-4. Loading of miRNA duplex into members of the Argonaute (Ago) protein family, representing the core of the RNA-induced silencing complex (RISC), is pivotal to miRNA-mediated gene silencing5-7. The Integrator complex has been previously shown to be an important regulator of RNA maturation, RNA polymerase II pause-release, and premature transcriptional termination8-11. Here, we report that loss of Integrator results in global diminution of mature miRNAs. By incorporating 4-Thiouridine (s4U) in nascent transcripts, we traced miRNA fate from biogenesis to stabilization and identified Integrator to be essential for proper miRNA assembly into RISC. Enhanced UV crosslinking and immunoprecipitation (eCLIP) of Integrator confirms a robust association with mature miRNAs. Indeed, Integrator potentiates Ago2-mediated cleavage of target RNAs. These findings highlight an essential role for Integrator in miRNA abundance and RISC function.

molecular biology↗

bioRxiv2020

Human ORC/MCM density is low in active genes and correlates with replication time but does not solely define replication initiation zones

Eukaryotic DNA replication initiates during S phase from origins that have been licensed in the preceding G1 phase. Here, we compare ChIP-seq profiles of the licensing factors Orc2, Orc3, Mcm3, and Mcm7 with gene expression, replication timing and fork directionality profiles obtained by RNA-seq, Repli-seq and OK-seq. ORC and MCM are strongly and homogeneously depleted from transcribed genes, enriched at gene promoters, and more abundant in early-than in late-replicating domains. Surprisingly, after controlling these variables, no difference in ORC/MCM density is detected between initiation zones, termination zones, unidirectionally replicating and randomly replicating regions. Therefore, ORC/MCM density correlates with replication timing but does not solely regulate the probability of replication initiation. Interestingly, H4K20me3, a histone modification proposed to facilitate late origin licensing, was enriched in late replicating initiation zones and gene deserts of stochastic replication fork direction. We discuss potential mechanisms that specify when and where replication initiates in human cells.

cell biology↗

bioRxiv2019

Active transcription regulates ORC/MCM distribution whereas replication timing correlates with ORC density in human cells

Eukaryotic replication initiates during S phase from origins that have been licensed in the preceding G1 phase. Here, we compare ChIP-seq profiles of the licensing factors Orc2, Orc3, Mcm3, and Mcm7 with replication initiation events obtained by Okazaki fragment sequencing. We demonstrate that MCM is displaced from early replicating, actively transcribed gene bodies, while ORC is mainly enriched at active TSS. Late replicating, H4K20me3 containing initiation zones display enhanced ORC and MCM levels. Furthermore, we find early RTDs being primarily enriched in ORC, compared to MCM, indicating that ORC levels are involved in organizing the temporal order of DNA replication. The organizational connection between active transcription and replication competence directly links changes in the transcriptional program to flexible replication patterns, which ensures the cells flexibility to respond to environmental cues.

cell biology↗