Maternal and zygotic gene regulatory effects of endogenous RNAi pathways

Abstract

11Endogenous small RNAs (sRNAs) and Argonaute proteins are ubiquitous regulators of gene 12 expression in germline and somatic tissues. sRNA-Argonaute complexes are often expressed in 13 gametes and are consequently inherited by the next generation upon fertilization. In Caenorhabditis 14 elegans, 26G-RNAs are primary endogenous sRNAs that trigger the expression of downstream 15 secondary sRNAs. Two subpopulations of 26G-RNAs exist, each of which displaying strongly 16 compartmentalized expression: one is expressed in the spermatogenic gonad and associates with the 17 Argonautes ALG-3/4; plus another expressed in oocytes and in embryos, which associates with the 18 Argonaute ERGO-1. The determinants and dynamics of gene silencing elicited by 26G-RNAs are 19 largely unknown. Here, we provide diverse new insights into these endogenous sRNA pathways of C. 20 elegans. Using genetics and deep sequencing, we dissect a maternal effect of the ERGO-1 branch 21 sRNA pathway. We find that maternal primary sRNAs can trigger the production of zygotic secondary 22 sRNAs that are able to silence targets, even in the absence of zygotic primary triggers. Thus, the 23 interaction of maternal and zygotic sRNA populations, assures target gene silencing throughout 24 Author Summary 31Small RNAs (sRNAs) and their partner Argonaute proteins regulate the expression of target 32RNAs. When sperm and egg meet upon fertilization, a diverse set of proteins and RNA, including 33 sRNA-Argonaute complexes, is passed on to the developing progeny. Thus, these two players are 34 important to initiate specific gene expression programs in the next generation. The nematode 35Caenorhabditis elegans expresses several classes of sRNAs. 26G-RNAs are a particular class of sRNAs 36 that are divided into two subpopulations: one expressed in the spermatogenic gonad and another 37 expressed in oocytes and in embryos. In this work, we describe the dynamics whereby oogenic 26G-38RNAs setup gene silencing in the next generation. We also show several ways that spermatogenic 39 26G-RNAs and their partner Argonautes, ALG-3 and ALG-4, use to regulate their targets. Finally, we 40show that ALG-3 and ALG-4 are fine-tuning their own expression, a rare role of Argonaute proteins. 41Overall, we provide new insights into how sRNAs and Argonautes are regulating gene expression. 42 (TEs). 48MicroRNA (miRNA), Piwi-interacting RNA (piRNA) and endogenous small interfering RNA 49 (endo-siRNA) pathways are the better described RNAi-like pathways, which differ in their biogenesis 50 and specialized cofactors. MicroRNAs are commonly found in many, if not all, tissues and broadly 51 regulate gene expression throughout development (1). piRNAs are typically, but not exclusively, 52 expressed in the metazoan germline, where they assume a central function in TE control (2-5). Endo-53 siRNA pathways comprise varied classes of sRNAs expressed in the soma and germline that can, for 54 example, control TEs, protein-coding genes and direct heterochromatin formation (6-8). A key 55 commonali...

Function and Evolution of Nematode RNAi Pathways

“…Interestingly, the regulatory effects of ALG-3/4 on their targets seem to be complex and dependent on temperature. At normal temperatures, regulation of ALG-3/4 targets seems to be predominantly repressive [30,79]. However, ALG-3/4 appear to promote gene expression at elevated temperatures, in the male germline [77].…”

“…Other than temperature, there are additional predictors of the regulatory outcome of ALG-3/4 targeting. Strikingly, ALG-3/4 branch 26G-RNAs preferentially originate from the 5′ and 3′ terminal regions of target transcripts [30,79]. Individual ALG-3/4 targets can have predominant 26G-RNA peaks on the 5′ or on the 3′ terminal regions.…”

The double-stranded DNA-binding proteins TEBP-1 and TEBP-2 form a telomeric complex with POT-1

“…To explore the expression pattern of tebp-1 and tebp-2 throughout animal development, we used a recently published mRNA-seq dataset (Almeida et al 2019). Both genes show the highest expression in embryos, very low abundance during the L1-L3 larval stages, and an increase in expression in L4 larvae and young adults (YAs, Supplemental Fig.…”

“…RNAseq data was taken from a previously published data set (Almeida et al 2019). To probe expression of the telomeric genes in spermatogenic and oogenic gonads (…”

“…RNA was extracted as described (Almeida et al 2019). Synchronized young adult animals were frozen in 50-100 µl of H2O after harvest.…”