An alternative spliceosome defined by distinct snRNAs in early zebrafish embryogenesis

Splicing removes intronic RNA sequences are removed from pre-mRNA molecules and enables, by alternative splicing, the generation of multiple unique RNA molecules from a single gene. As such, splicing is an essential part of the whole translation system of a cell. The spliceosome is a ribonucleoprotein complex in which five small nuclear RNAs (snRNAs) are involved; U1, U2, U4, U5, and U6. For each of these snRNAs there are variant gene copies present in a genome. Furthermore, in many eukaryotic species there is an alternative, minor spliceosome that can splice a small number of specific introns. As we previously discovered an embryogenesis-specific ribosomal system in zebrafish early embryogenesis based on variant rRNA and snoRNA expression, we hypothesized that there may also be an embryogenesis-specific spliceosome. An inventory of zebrafish snRNA genes revealed clustered and dispersed loci for all but U2 major snRNAs. For each minor spliceosome snRNA, just one gene locus was found. Since complete snRNA molecules are hard to sequence, we employed a combined PCR-sequencing approach to measure the individual snRNA-variant presence. Analysis of egg and male-adult samples revealed embryogenesis-specific and somatic-specific variants for each major snRNA. These variants have substantial sequence differences, yet none in their mRNA binding sites. Given that many of the sequence differences are found in loop structures indicate possible alternative protein binding. Altogether, with this study we established that the spliceosome is also an element of the embryogenesis-specific translation system in zebrafish.


INTRODUCTION
29 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 snRNA genes are present within the same organisms (5-9).
43 This raises the question why these variants exist and what role they might play in 44 translation. Although expression of these variants has been extensively studied, there is still 45 not a clear understanding for the existence of these snRNA variants (10). 53 in human (6). snRNAs also display a tissue-preferred expression, which implicate them in 54 different disease pathologies such as several neurological diseases (7,10,23,24). 55 The fact that there are snRNAs which variants are differentially expressed during 56 embryogenesis relates to our previous findings where we discovered distinct maternal-types 57 of rRNAs and snoRNAs specifically expressed during early zebrafish embryogenesis (25).
58 These maternal-type RNAs seem to be part of a distinct early embryogenesis-specific 88 unannotated major and minor spliceosome snRNA loci. This resulted in a total of 958 snRNA 89 loci (Table 1, Supplemental Table ST1  110 After cataloguing all snRNAs in the zebrafish genome, we investigated whether 111 embryogenesis-specific snRNAs exist by examining their expression in eggs and somatic 145 nucleotide sequences that bind to the mRNA, thus allowing each system to splice distinct 146 introns. However, even though there are many sequence differences between the maternal-147 type and the somatic-type snRNAs, none of them involve the mRNA binding sites in these 148 snRNAs (Figure 2 and Supplement File SF3). It turned out that the snRNA sequence 149 differences are often located in specific parts of the secondary structure (Figure 2). For 150 instance, for U1, all but one differences are located in one stem-loop and in U2 they are 151 confined to one side of the structure (Figure 2). In general, many of the differences are 152 found in the loops, which are thought to be specific binding locations for spliceosomal 153 proteins. Hence, this would indicate that the embryogenesis spliceosome, besides specific 154 snRNAs also may comprise (embryogenesis-)specific proteins.
155 Despite many apparently co-evolved nucleotide pairs in stems of the snRNAs (Figure 2 258 RT-PCR-qSeq analysis 259 Forward and reverse PCR primers were designed for maternal-type and somatic-type snRNA 260 variants, in such a way that: 1) as much as possible of the 5'-end of the full-length variants is 261 included in the final amplicon, and 2) generic primers will bind to the maternal-type, as well 262 as the somatic-type variants (Supplemental Table ST4 and Figure 1A). To avoid positive 263 results due to genomic DNA background, small RNA-enriched total RNA was treated twice 264 with 5 l of RNase-free DNase (Qiagen) for 45 minutes at 37°C. Next, cDNA was prepared