An alternative spliceosome defined by distinct snRNAs in early zebrafish embryogenesis

Abstract

Alternative snRNAs in early zebrafish embryogenesissnRNA, spliceosome, dual translation machinery, embryogenesis 2 1 ABSTRACT 2 Splicing removes intronic RNA sequences are removed from pre-mRNA molecules and 3 enables, by alternative splicing, the generation of multiple unique RNA molecules from a 4 single gene. As such, splicing is an essential part of the whole translation system of a cell. 5 The spliceosome is a ribonucleoprotein complex in which five small nuclear RNAs (snRNAs) 6 are involved; U1, U2, U4, U5, and U6. For each of these snRNAs there are variant gene 7 copies present in a genome. Furthermore, in many eukaryotic species there is an 8 alternative, minor spliceosome that can splice a small number of specific introns. As we 9 previously discovered an embryogenesis-specific ribosomal system in zebrafish early 10 embryogenesis based on variant rRNA and snoRNA expression, we hypothesized that there 11 may also be an embryogenesis-specific spliceosome. An inventory of zebrafish snRNA genes 12 revealed clustered and dispersed loci for all but U2 major snRNAs. For each minor 13 spliceosome snRNA, just one gene locus was found. Since complete snRNA molecules are 14 hard to sequence, we employed a combined PCR-sequencing approach to measure the 15 individual snRNA-variant presence. Analysis of egg and male-adult samples revealed 16 embryogenesis-specific and somatic-specific variants for each major snRNA. These variants 17 have substantial sequence differences, yet none in their mRNA binding sites. Given that 18 many of the sequence differences are found in loop structures indicate possible alternative 19 protein binding. Altogether, with this study we established that the spliceosome is also an 20 element of the embryogenesis-specific translation system in zebrafish. 21 22 23 24 25 26 27 3 28 INTRODUCTION29 Alternative splicing is fundamental for gene regulation and the generation of different 30 transcripts and/or proteins from an individual gene in eukaryotes (1). Splicing is executed by 31 the spliceosome and removes intronic sequences from pre-mRNA during the maturation 32 process in which the exonic sequences eventually form the mRNA (2,3).The spliceosome is a 33 molecular complex formed by hundreds of proteins and five essential small-nuclear RNAs 34 (snRNAs) that are typically located in the nucleus. The size of these small RNA molecules 35 ranges from 118 nucleotides (nt) to 191 nt. As they are uracil rich, they are called U1, U2, 36 U4, U5 and U6 snRNAs. Next to this major spliceosome, a minor (or U12 dependent) 37 spliceosome exists in many eukaryotic species, which is involved in the splicing of a relative 38 small number of specific introns (4). The snRNAs involved in the minor spliceosome are: 39 U11, U12, U4atac, and U6atac, completed by the U5 from the major spliceosome (4).40 As splicing is at the core of the cellular translation system, the sequences of the involved 41 snRNA are highly conserved across species. At the same time, many non-canonical variants 42 and gene copies of the major s...

New observations on non-coding RNAs involved in the dual translation system in zebrafish development

“…In this manuscript, we presented the preliminary results of our initial gene-expression analyses of the non-coding RNA types involved in the zebrafish translation system, other than we previously reported on (Locati et al 2017a(Locati et al , 2017b(Locati et al , 2018Pagano et al 2019bPagano et al , 2019a. Alike rRNA (Locati et al 2017a(Locati et al , 2017b, snoRNA (Pagano et al 2019b), and snRNA (Pagano et al 2019a), the investigated tRNA, RNase-P and SRP-RNA all seem to have a maternal-specific variant. Together, a complete maternal-specific translation system emerges, which is dedicated to oogenesis and (early) embryogenesis in zebrafish.…”

“…This maternaltype 5S RNA variant is gradually replaced throughout embryonic development by a somatictype variant (Wegnez et al 1972;Brown et al 1977;Wormington and Brown 1983;Guinta et al 1986;Komiya et al 1986). Starting from this long-standing observation, our continuous studies reported in several manuscripts (Locati et al 2017a(Locati et al , 2017b(Locati et al , 2018Pagano et al 2019bPagano et al , 2019a show the existence of a specialized maternal-type translation system in zebrafish far beyond just 5S rRNA. One by one, we have revealed that also the translation-involved non-coding RNAs 5.8S, 18S, 28S, snoRNA, and snRNA are implicated in this maternal-type translation system ( Figure 1 and Table 1).…”

“…For almost all investigated anticodon tRNAs, expression data was obtained by analyzing egg and adult male tail samples (cf. M&M (Pagano et al 2019a)). An initial analysis clearly showed that there is differential expression between different isodecoders of each analyzed anticodon tRNA ( Figure 2C and 2D).…”

“…We therefore employed our RT-PCR-qSeq approach (cf. M&M (Pagano et al 2019a)) with PCR primer pairs hybridizing to the 20 most 5' terminal and 3' terminal bases of nearly all anticodon tRNAs ( Supplemental Table ST1). Regrettably, this approach leaves only about 40 nucleotides for isodecoder identification.…”

Immunoglobulin switch-like recombination regions implicated in the formation of extrachromosomal circular 45S rDNA involved in the maternal-specific translation system of zebrafish

“…Over the last years, we have reported on the discovery of an intriguing dual translation system in zebrafish development (Locati et al 2017a(Locati et al , 2017b(Locati et al and 2018Pagano et al 2019a and2019b;Breit et al 2019). The two systems encompass at least all essential non-coding RNA containing components of the translation process: the spliceosomes (snRNA) (Pagano et al 2019b); the ribosomes (5S, 5.8S, 18S, and 28S rRNA) (Locati et al 2017a(Locati et al , 2017b(Locati et al and 2018 plus the pre-rRNA processing snoRNAs (Pagano et al 2019a); the tRNAs (Breit et al 2019) plus the pre-tRNA processing RNaseP (Breit et al 2019); and the Signal Recognition Particle (SRP-RNA) for translocation of the translational complex to the ER (Breit et al 2019). Of this dual translation system, the maternal-type variant (45S-M) is produced during oogenesis and progressively replaced by the somatic-type variant (45S-S) during embryogenesis (Locati et al 2017b, Ortega-Recalde et al 2019.…”