Biology subjects

Kanhere, A.

Publications and source records attributed to Kanhere, A..

6 recordsLinked to original sources

bioRxiv2026

PI3K-AKT activation determines oncogenic RAS-induced hypertranscription and replication stress

Hypertranscription and transcription-replication conflicts (TRCs) are frequent features of cancer cells. RAS oncogenes promote hypertranscription to allow cell growth and proliferation, which can the lead to TRCs. Here, we report that hyperactivation of the PI3K-AKT signalling pathway is required for TRCs induced by RAS oncogenes. Oncogenic HRAS causes more TRCs than oncogenic KRAS or BRAF, because HRAS hyperactivates PI3K. PI3K hyperactivation is associated with in glycogen synthase kinase-3{beta} (GSK3{beta}) inhibition, increased E2F and MYC transcription programmes, increased nascent transcription of ribosome biogenesis genes and small nucleolar RNAs (snoRNA) expression. Small molecule inhibition of PI3K signalling prevents RAS-induced replication stress, and small molecule PI3K activation promotes replication stress. RAS-induced TRCs require a cooperation of MAPK and Pi3K signalling, S phase entry and hypertranscription. Our findings suggest a mechanistic explanation for replication stress variability between RAS activation models and identify PI3K pathway activation as a potential new determinant of TRCs in cancer.

cancer biology↗

bioRxiv2025

The 7SK small nuclear ribonucleoprotein links the cell responses to transcription and replication stress by promoting replication fork reversal and homologous recombination

The 7SK-small nuclear ribonucleoprotein complex (7SK-snRNP) plays a crucial role in the response to transcription stress, releasing positive transcription elongation factor P-TEFb to sustain RNA polymerase II activity when transcription is blocked. Many conditions that block transcription also block DNA replication, causing replication stress, and 7SK-snRNP components are putative tumour suppressors with roles in transcription-replication conflicts and double-strand break repair. Here, we investigate potential roles of 7SK-snRNP components in the response to replication stress induced by chemotherapy agents with and without additional impact on transcription. We report that HEXIM1 and LARP7 promote replication fork slowing in response to agents that cause both replication- and transcription stress, in a manner consistent with their canonical 7SK-snRNP functions. Our data suggest that this role in fork slowing is mainly through facilitating RAD51-mediated replication fork reversal rather than transcription-replication conflicts. HEXIM1 and LARP7 promote RAD51 recruitment to replication-associated DSBs or post-replicative gaps under conditions of transcription stress such as induced by camptothecin or BET inhibitors and support HR at direct DSBs. In contrast, HEXIM1 and LARP7 are not required for HR in response to hydroxyurea, which does not cause transcription stress. Our data support that LARP7 roles during replication stress are independent of its reported interaction with BRCA1, and that both LARP7 and HEXIM1 promote survival in response to replication stress-inducing agents. 7SK-snRNP components are not recruited to stressed replication forks and RNA polymerase II inhibition phenocopies loss of these proteins. Taken together, our data support a model where 7SK-snRNP modulation of RNA polymerase II activity helps facilitate RAD51 function under transcription stress conditions, thereby connecting the cell responses to transcription- and replication stress.

molecular biology↗

bioRxiv2024

Human RNase H2 upregulation counteracts oncogene- and chemotherapy-induced replication stress

RNase H2 is a heterotrimeric endoribonuclease that resolves RNA:DNA hybrids and genome-embedded ribonucleotides, which are implicated in DNA replication stress and cancer development. Protein and/or mRNA levels of individual RNase H2 subunits are elevated in some cancers, but little is known about the mechanisms or consequences of RNase H2 upregulation. We report that RNase H2 subunits are upregulated at the protein level in response to replication stress induced by oncogenes and chemotherapy drugs in human cancer and non-cancer cell lines. We show that inducible overexpression of the RNASEH2B subunit increases levels of the active RNase H2 heterotrimer. While causing only subtle changes to gene expression, RNASEH2B overexpression is unexpectedly associated with increased RNA:DNA hybrid levels. RNASEH2B overexpression prevents further increases in RNA:DNA hybrid levels by camptothecin or hydroxyurea and reduces replication fork stalling in presence of these drugs. Surprisingly, RNase H2 levels do not strongly impact survival of chemotherapy treatments but appear to have more subtle effects on genome instability and innate immune signalling. In contrast, increased RNase H2 levels in presence of oncogenic HRAS limit not only RAS-induced replication fork stalling but also cell death. Our findings shed new light on the functions of RNase H2 and suggest that upregulation of RNase H2 may be an important aspect of replication stress responses in cancer.

molecular biology↗

bioRxiv2024

Investigating the role of CpG island DNA methylation at 3'UTRs in cancer

DNA methylation is one of the most important epigenetic processes that regulates gene expression. While the human genome is predominately methylated, CpG-rich regions known as CpG islands (CGIs) are unmethylated and are sites of transcriptional control. While the importance of CGI and DNA methylation at gene promoters is well understood, the significance of CGIs at 3 untranslated regions (3UTRs) remains largely unexplored. In this study, we characterised CGIs located within 3UTRs and investigated their role in gene regulation. Here, we show that around 3% of CGIs (909 CGIs) are exclusively located at 3UTR and are not associated with any nearby promoters. Importantly, 3UTR CGIs are highly conserved, with a conservation score even higher than promoter CGI implying their importance. In contrast to promoter CGIs which are predominantly unmethylated, 3UTR CGIs are often methylated. Genes with 3UTR CGIs are associated with several different cancers and cancer-related signalling pathways. Moreover, 3UTR CGIs are differentially methylated in cancers, with changes in methylation associated with changes in gene expression. Together this data suggests the importance of 3UTR CGIs and their methylation in gene expression regulation in cancer.

genomics↗

bioRxiv2020

A long intergenic non-coding RNA regulates nuclear localisation of DNA methyl transferase-1

DNA methyl-transferase-1 or DNMT1 maintains DNA methylation in the genome and is important for regulating gene expression in cells. Aberrant changes in DNMT1 activity are observed in many diseases. Therefore, understanding the mechanisms behind alteration of DNMT1 activity is important. Here, we show that CCDC26, a nuclear long non-coding RNA frequently mutated in myeloid leukaemia, directly interacts with DNMT1. In the absence of CCDC26 RNA, DNMT1 is mis-located in the cytoplasm. As a result, genomic DNA is significantly hypomethylated, which is accompanied by a slower cell growth rate and increased cell death. These results point to a previously unrecognised mechanism of long non-coding RNA mediated subcellular localisation of DNMT1 and regulation of DNA methylation. These observations are significant given the importance of DNMT1 in cancer and number of other diseases.

molecular biology↗

bioRxiv2020

Antisense ncRNAs during early vertebrate development are divided in groups with distinct features

Long non-coding RNAs or lncRNAs are a broad class of non-protein coding RNAs that are >200nucleotides in length. A number of lncRNAs are shown to play an important role in gene expression regulation. LncRNAs antisense to a protein-coding gene can act either as positive or negative regulators of overlapping protein-coding mRNAs. Almost 50% of lncRNAs present during development of vertebrates such as zebrafish are of antisense lncRNA class. However, their role in gene expression regulation during development remains enigmatic. To understand the role of antisense lncRNAs in early vertebrate development, we took a computational biology approach to analyze existing as well as novel dataset. Our analysis of RNA sequencing data from zebrafish development indicates that antisense RNAs can be divided into two major classes based on their positive or negative co-expression patterns to the sense protein-coding genes. The ones with negative co-expression patterns or group-1 are maternal antisense lncRNAs that overlap mainly developmental genes. Group-2 with positive expression pattern overlap mainly house-keeping genes. Group-1 antisense lncRNAs are longer and more stable as compared to antisense lncRNAs in group-2. In addition, to answer if antisense RNAs in the two groups are differently localized in cell compartments, we deep-sequenced RNA from cytoplasmic and nuclear compartments during early developmental stages. The analysis of these compartment specific datasets revealed group-1 lncRNAs are cytosolic. Based on the cytosolic nature of group-1 RNAs and their higher complementarity to the overlapping developmental mRNAs, we speculate that the group-1 RNAs might function similar to microRNAs in silencing spurious expression of developmental genes. Group-1 and group-2 RNAs are also distinct in terms of their genomic configuration, conservation, length and transcriptional regulation. These results are not only important in understanding the role of antisense RNAs in development but also for predicting the nature of association between antisense lncRNA and overlapping protein-coding genes.

genomics↗