Biology subjects

Knittel, T. L.

Publications and source records attributed to Knittel, T. L..

3 recordsLinked to original sources

bioRxiv2024

Regulation of Microprocessor assembly and localization via Pasha's WW domain in C. elegans

Primary microRNA (pri-miRNA) transcripts are processed by the Microprocessor, a protein complex that includes the ribonuclease Drosha and its RNA binding partner DGCR8/Pasha. We developed a live, whole animal, fluorescence-based sensor that reliably monitors pri-miRNA processing with high sensitivity in C. elegans. Through a forward genetic selection for alleles that desilence the sensor, we identified a mutation in the conserved G residue adjacent to the namesake W residue of Pashas WW domain. Using genome editing we also mutated the W residue and reveal that both the G and W residue are required for dimerization of Pasha and proper assembly of the Microprocessor. Surprisingly, we find that the WW domain also facilitates nuclear localization of Pasha, which in turn promotes nuclear import or retention of Drosha. Furthermore, depletion of Pasha or Drosha causes both components of the Microprocessor to mislocalize to the cytoplasm. Thus, Pasha and Drosha mutually regulate each others spatial expression in C. elegans.

molecular biology↗

bioRxiv2024

A low-abundance class of Dicer-dependent siRNAs produced from a variety of features in C. elegans

Canonical small interfering RNAs (siRNAs) are processed from double-stranded RNA (dsRNA) by the endoribonuclease Dicer. siRNAs are found in plants, animals, and some fungi where they associate with Argonautes to direct RNA silencing. In Caenorhabditis elegans, some endogenous small RNAs, such as 22G-RNAs and 26G-RNAs, share certain attributes with canonical siRNAs but exhibit unique characteristics known only to occur in nematodes. For instance, 22G-RNAs do not originate from dsRNA and are not processed by Dicer, whereas 26G-RNAs require Dicer but lack the typical duplex intermediate with symmetrical 3-overhangs and are produced only antisense to their mRNA templates. To identify canonical siRNAs in C. elegans, we first characterized the siRNAs produced from exogenous dsRNA. As predicted based on earlier studies, exogenous dsRNA is processed into [~]23-nt duplexes with 2-4-nt 3-overhangs, ultimately yielding siRNAs devoid of 5 G-containing sequences that bind with high affinity to the Argonaute RDE-1. Leveraging these characteristics, we searched for their endogenous counterparts and identified thousands of endogenous loci representing dozens of unique elements that give rise to mostly low to moderate levels of siRNAs, called 23H-RNAs. These loci include repetitive elements, alleged coding genes, pseudogenes, non-coding RNAs, and unannotated features, many of which adopt hairpin structures reminiscent of the hpRNA/RNA interference (RNAi) pathway in flies and mice. Our results expand the known repertoire of C. elegans small RNAs and demonstrate that key features of the endogenous siRNA pathway are relatively unchanged in animals.

genomics↗

bioRxiv2022

Tissue-specific overexpression of the double-stranded RNA transporter SID-1 limits lifespan in C. elegans

Intertissue RNA transport has emerged as a novel signaling mechanism. In C. elegans, this is conferred by the systemic RNAi pathway, in which the limiting step is the cellular import of extracellular RNAs via SID-1. To better understand the physiological role of systemic RNAi in vivo, we modified the function of SID-1 through loss-of-function mutation and tissue-specific overexpression of sid-1 in C. elegans. We observed that sid-1 loss-of-function mutants are as healthy as wild-type worms. Conversely, overexpression of sid-1 in intestine, muscle, or neurons rendered worms short-lived. The effects of intestinal sid-1 overexpression were reversed by silencing the components of the systemic RNAi pathway sid-1, sid-2 and sid-5, thus implicating RNA transport. Moreover, silencing the miRNA biogenesis proteins pash-1 and dcr-1 rendered the lifespan of worms with intestinal sid-1 overexpression similar to controls. Lastly, we observed that the lifespan decrease produced by tissue-specific sid-1 overexpression was dependent on the bacterial food source. Collectively, our data support the notion that systemic RNA signaling is tightly regulated, and unbalancing that process provokes a reduction in lifespan.

molecular biology↗