Expression of of distinct maternal and somatic 5.8S, 18S and 28S rRNA types during zebrafish development

Animal science

Identifying small RNAs derived from maternal- and somatic-type rRNAs in zebrafish development

rRNAs are non-coding RNAs present in all prokaryotes and eukaryotes. In eukaryotes there are four rRNAs: 18S, 5.8S, 28S, originating from a common precursor (45S), and 5S. We have recently discovered the existence of two distinct developmental types of rRNA: a maternal-type, present in eggs and a somatic-type, expressed in adult tissues. Lately, next-generation sequencing has allowed the discovery of new small-RNAs deriving from longer non-coding RNAs, including small-RNAs from rRNAs (srRNAs). Here, we systemically investigated srRNAs of maternal- or somatic-type 18S, 5.8S, 28S, with small-RNAseq from many zebrafish developmental stages. We identified new srRNAs for each rRNA. For 5.8S, we found srRNA consisting of the 5' or 3' halves, with only the latter having different sequence for the maternal- and somatic-types. For 18S, we discovered 21 nt srRNA from the 5' end of the 18S rRNA with a striking resemblance to microRNAs; as it is likely processed from a stem-loop precursor and present in human and mouse Argonaute-complexed small-RNA. For 28S, an abundant 80 nt srRNA from the 3' end of the 28S rRNA was found. The expression levels during embryogenesis of these srRNA indicate they are not generated from rRNA degradation and might have a role in the zebrafish development

Improving small RNA-seq by using a synthetic spike-in set for size-range quality control together with a set for data normalization

There is an increasing interest in complementing RNA-seq experiments with small-RNA (sRNA) expression data to obtain a comprehensive view of a transcriptome. Currently, two main experimental challenges concerning sRNA-seq exist: how to check the size distribution of isolated sRNAs, given the sensitive size-selection steps in the protocol; and how to normalize data between samples, given the low complexity of sRNA types. We here present two separate sets of synthetic RNA spike-ins for monitoring size-selection and for performing data normalization in sRNA-seq. The size-range quality control (SRQC) spike-in set, consisting of 11 oligoribonucleotides (10-70 nucleotides), was tested by intentionally altering the size-selection protocol and verified via several comparative experiments. We demonstrate that the SRQC set is useful to reproducibly track down biases in the size-selection in sRNA-seq. The external reference for data-normalization (ERDN) spike-in set, consisting of 19 oligoribonucleotides, was developed for sample-to-sample normalization in differential-expression analysis of sRNA-seq data. Testing and applying the ERDN set showed that it can reproducibly detect differential expression over a dynamic range of 218. Hence, biological variation in sRNA composition and content between samples is preserved while technical variation is effectively minimized. Together, both spike-in sets can significantly improve the technical reproducibility of sRNA-seq

Maternal- and Somatic-type snoRNA Expression and Processing in Zebrafish Development

Small nucleolar RNAs (snoRNAs) are non-coding RNAs that play an important role in the complex maturation process of ribosomal RNAs (rRNAs). SnoRNAs are categorized in classes, with each class member having several variants present in a genome. Similar to our finding of specific rRNA expression types in zebrafish embryogenesis, we discovered preferential maternal- and somatic-expression for snoRNAs. Most snoRNAs and their variants have higher expression levels in somatic tissues than in eggs, yet we identified three snoRNAs; U3, U8 and snoZ30 of which specific variants show maternal- or somatic-type expression. For U3 and U8 we also found small-derived snoRNAs that lack their 5’ rRNA recognition part and are essentially Domain II hairpin structures (U-DII). These U-DII snoRNAs from variants showed similar preferential expression, in which maternal-type variants are prominently expressed in eggs and subsequently replaced by a somatic-type variants during embryogenesis. This differential expression is related to the organization in tandem repeats (maternal type) or solitary (somatic-type) genes of the involved U snoRNA loci. The collective data showed convincingly that the preferential expression of snoRNAs is achieved by transcription regulation, as well as through RNA processing. Finally, we observed small-RNAs derived from internal transcribed spacers (ITSs) of a U3 snoRNA loci that via complementarity binding, may be involved in the biosynthesis of U3-DII snoRNAs. Altogether, the here described maternal- and somatic-type snoRNAs are the latest addition to the developing story about the dual ribosome system in zebrafish development

Linking Maternal and Somatic 5S rRNA types with Different Sequence-Specific Non-LTR Retrotransposons

Animal science