Systematic, RNA-Interference-Mediated Identification of mus-101 Modifier Genes in Caenorhabditis elegans

Abstract

The Mus101 family of chromosomal proteins, identified initially in Drosophila, is widely conserved and has been shown to function in a variety of DNA metabolic processes. Such functions include DNA replication, DNA damage repair, postreplication repair, damage checkpoint activation, chromosome stability, and chromosome condensation. Despite its conservation and widespread involvement in chromosome biogenesis, very little is known about how Mus101 is regulated and what other proteins are required for Mus101 to exert its functions. To learn more about Mus101, we have initiated an analysis of the protein in C. elegans. Here, we show that C. elegans mus-101 is an essential gene, that it is required for DNA replication, and that it also plays an important role in the DNA damage response. Furthermore, we use RNA interference (RNAi)-mediated reverse genetics to screen for genes that modify a mus-101 partial loss-of-function RNAi phenotype. Using a systematic approach toward modifier gene discovery, we have found five chromosome I genes that modify the mus-101 RNAi phenotype, and we go on to show that one of them encodes an E3 SUMO ligase that promotes SUMO modification of MUS-101 in vitro. These results expand our understanding of MUS-101 regulation and show that genetic interactions can be uncovered using screening strategies that rely solely on RNAi.

Cell cycle progression requires the CDC-48 UFD−1/NPL−4 complex for efficient DNA replication

“…In contrast, in cdc-48(RNAi), ufd-1(RNAi), and npl-4(RNAi) embryos the separating chromatids often remain connected via chromosome bridges. This phenotype is reported to be associated with S phase defects (17,23), and occurs throughout the divisions of P 0 , AB, and P1 cells (Fig. 1E, Fig.…”

In vivo and in vitro knockdown of FREP2 gene expression in the snail Biomphalaria glabrata using RNA interference

“…Most in vivo RNAi studies have been performed with invertebrates, mainly arthropods, with the aim of directly assessing phenotype changes. These invertebrates include Caenorhabditis elegans [9,10], digenetic trematodes [11,12], silkworms [13], moth Spodoptera liturs [14], Drosophila [15][16][17], mosquitoes [18,19], honeybee [20], and ticks [21]. In vitro RNAi assays have also been widely applied to cell lines from humans and model organisms such as C. elegans [10], Drosophila [22,23], mosquitoes [24], and mice [25,26].…”

Using RNA Interference to Identify Specific Modifiers of a Temperature-Sensitive, Embryonic-Lethal Mutation in the Caenorhabditis elegans Ubiquitin-Like Nedd8 Protein Modification Pathway E1-Activating Gene rfl-1

“…Another strong specific suppressor, called mus-101, encodes a widely conserved 1227-amino-acid protein containing six copies of the BRCA1 carboxyl-terminal (BRCT) repeat (Yamamoto et al 2000;Holway et al 2005). The mus-101 gene is essential but feeding RNAi results in only partially penetrant embryonic lethality (Maeda et al 2001;Holway et al 2005).…”

“…The mus-101 gene is essential but feeding RNAi results in only partially penetrant embryonic lethality (Maeda et al 2001;Holway et al 2005). BRCT domains are commonly found in proteins involved in DNA metabolism and have been implicated in mediating protein-protein interactions between other BRCT domain-containing proteins (Manke et al 2003;Yu et al 2003).…”