Biology subjects

Singh, J.

Publications and source records attributed to Singh, J..

3 recordsLinked to original sources

bioRxiv2018

Cdc23/Mcm10 Primase Generates the Lagging Strand-Specific Ribonucleotide Imprint in Fission Yeast

The developmental asymmetry of fission yeast daughter cells derives from inheriting \"older Watson\" versus \"older Crick\" DNA strand from the parental cell, strands that are complementary but not identical with each other. A novel DNA strand-specific \"imprint\", installed during DNA replication at the mating-type locus (mat1), imparts competence for cell type inter-conversion to one of the two chromosome replicas. The biochemical nature of the imprint and the mechanism of its installation are still not understood. The catalytic subunit of DNA Polymerase (Pol) has been implicated in the imprinting process. Based on its known biochemical function, Pol might install the mat1 imprint during lagging strand synthesis. The nature of the imprint is not clear: it is either a nick or a ribonucleotide insertion. Our investigations do not support a role of Pol in nicking through putative endonuclease domains but confirm its role in installing an alkali-labile moiety as the imprint. A detailed genetic and molecular analysis reveals a direct role of the Cdc23/Mcm10 primase activity in installing the imprint in cooperation with Pol and Swi1.

genetics↗

bioRxiv2017

siRNA biogenesis, DNA methylation and target locus silencing are distinct and separable Pol IV sub-functions differentially dependent on the largest subunit's CTD

Plant nuclear multisubunit RNA polymerase IV plays a key role in the RNA-directed DNA methylation (RdDM) pathway for transcriptional silencing of transposons, viruses and specific genes by synthesizing precursors of 24 nt siRNAs that guide the process. The Pol IV largest subunit, NRPD1 is derived from the Pol II largest subunit but has a unique carboxy-terminal domain (CTD) of unknown function. We show that the NRPD1 CTD is critical for transcriptional silencing of target loci and for producing 24 nt siRNAs at high levels. However, the CTD is surprisingly dispensable for near wild-type levels of Pol IV-dependent genomic cytosine methylation. These results suggest that low levels of 24 nt siRNAs, produced at only 20-30% of wild-type levels, are sufficient for full RNA-directed DNA methylation, yet insufficient for silencing, suggesting additional roles for siRNAs beyond DNA methylation. Moreover, at a subset of target loci, neither siRNA levels nor cytosine methylation are impaired upon deletion of the CTD, yet silencing is lost. Collectively, the non-linear relationships between siRNA levels, cytosine methylation and silencing suggest the existence of additional mechanisms of silencing dependent on Pol IV transcription and mediated by the CTD, such as promoter occlusion to inhibit the activities of other polymerases.

molecular biology↗

bioRxiv2017

Subdomains of the Pol V largest subunit CTD mediate locus-specific RNA-directed DNA methylation

Plant multisubunit RNA Polymerase V transcription recruits Argonaute siRNA complexes that specify sites of RNA-directed DNA methylation (RdDM) for gene silencing. Pol V's largest subunit, NRPE1, evolved from the largest subunit of Pol II but has a distinctive carboxyl-terminal domain (CTD). We show that the Pol V CTD is dispensable for catalytic activity in vitro, yet essential in vivo. One CTD subdomain (DeCL), is required for Pol V function at virtually all loci. other CTD subdomains have locusspecific effects. In a yeast two-hybrid screen, the 3'->5' exoribonuclease, RRP6L1 was identified as an interactor with the DeCL subdomain and DeCL and glutamine-serine-rich (QS) subdomains, located downstream from an Argonaute-binding repeat subdomain. Experimental evidence indicates that RRP6L1 trims the 3 ends of Pol V transcripts sliced by ARGONAUTE 4 (AGO4), suggesting a model whereby the CTD enables the spatial and temporal coordination of AGO4 and RRP6L1 RNA processing activities.

molecular biology↗