Transcriptome-wide analysis of small RNA expression in early zebrafish development

Abstract

During early vertebrate development, a large number of noncoding RNAs are maternally inherited or expressed upon activation of zygotic transcription. The exact identity, expression levels, and function for most of these noncoding RNAs remain largely unknown. miRNAs (microRNAs) and piRNAs (piwi-interacting RNAs) are two classes of small noncoding RNAs that play important roles in gene regulation during early embryonic development. Here, we utilized next-generation sequencing technology to determine temporal expression patterns for both miRNAs and piRNAs during four distinct stages of early vertebrate development using zebrafish as a model system. For miRNAs, the expression patterns for 198 known miRNAs within 122 different miRNA families and eight novel miRNAs were determined. Significant sequence variation was observed at the 5' and 3'ends of miRNAs, with most extra nucleotides added at the 3' end in a nontemplate directed manner. For the miR-430 family, the addition of adenosine and uracil residues is developmentally regulated and may play a role in miRNA stability during the maternal zygotic transition. Similar modification at the 3' ends of a large number of miRNAs suggests widespread regulation of stability during early development. Beside miRNAs, we also identified a large and unexpectedly diverse set of piRNAs expressed during early development.

FIGURE 1.

Sequencing summary. (A) Size distribution of all sequencing reads between 18 and 30 nt. RNA reads derived from four developmental stages are indicated in different colors. The size distribution and abundance of the reads from each stage are as indicated. (B) Read frequency for all sequences. The identity and frequency of small RNAs reads from different developmental time points are as indicated. (C) Read frequency for unique sequences. In contrast to B, where the total read frequencies were charted, small RNA reads derived from the same miRNA were grouped together as a single subset. This analysis shows that a large number of unique reads are derived from distinct genomic elements, mostly repetitive elements.

FIGURE 2.

miRNA family expression profiles. miRNA family read frequency was normalized (see Materials and Methods) and compared across four developmental stages. Yellow indicates high expression and blue indicates low expression. Gray indicates undetectable levels of expression.

FIGURE 3.

Northern blot of novel miRNAs. Northern blots were performed to verify the expression of predicted novel miRNAs. Mature miRNAs were detected for six of the eight predicted miRNAs, and miRNA precursors were detected for five of the six miRNAs.

FIGURE 4.

miRNA sequence heterogeneity. (A) Significant sequence variation was detected among mature miRNA reads, especially at 3′ ends. Shown are compilations from four different miRNAs with the extent of variation at any particular nucleotide indicated by the size of the font. (Below) In red is the mature miRNA sequence with adjacent genomic sequence in black. Nontemplate-directed 3′ additions are shown in color. (B) Similar composition of 3′ addition among different miRNAs of the same family. The extent and base composition of miRNA tailing is indicated for a representative subset of miRNA families at the indicated stages of development. The percentage of reads with different nucleotide 3′ ends is shown in different colors. (C) Modification of mature miR-430 reads. The ratio of both A- or U-tailed mature miR-430 family reads to total mature miR-430 reads is shown across four developmental stages. (D) Modification of miR-430 reads subject to trimming. A large number of miR-430 reads did not contain the normal terminal guanosine. For this subset, the ratio of A- or U-tailed RNAs miR-430 reads is shown across four developmental stages. (E) The percentage of U-tailed miRNA reads for all tailed miRNA reads in both 256-cell and sphere stage embryos. All miRNAs with 3′ additions of nontemplate-directed U residues are shown for the 256-cell and sphere stage. The percentage of U addition increased from the 256-cell stage to the sphere stage.

FIGURE 5.

Zebrafish piRNA expression. (A,B) After filtering out miRNA reads, the size distribution and abundance of reads mapping to unique (A) or repetitive (B) genomic loci is shown. Different colors represent reads from different developmental stages. (C) Correlation between the abundance of reads derived from shield stage small RNAs and the density of transposable elements along chromosome 11. At the top, small RNAs that map to chromosome 11 are indicated either as unweighted (gray), meaning the total number of reads irrespective of how many positions (copies) along a chromosome that might encode this RNA, or weighted (orange line), meaning the total number of reads divided by the number of positions or copies within the zebrafish genome. Transposable elements and transposons were divided into DNA/LTR (blue) and LINE/SINE (red) based on the origin of the small RNA reads. The density of repetitive elements (ratio) was determined by the percentage of nucleotides mapping to transposable elements per 50 kb. (D,E) Genomic localization of piRNA clusters. Vertical lines represent piRNA clusters from four developmental time points and also from data generated from adult ovaries and testes across either chromosome 4 or chromosome 5. Red lines indicate plus strands, while blue lines indicate minus strands. (F,G) Quantification of expression of piRNA clusters from total reads (F) or reads derived from unique genomic loci (G). Black and gray columns represent the strands from which the reads originated.

FIGURE 6.

Zebrafish tRNA-derived Fragments (tRF). (A) Graphic representation of tRF alignments to zebrafish tRNAs. Most reads mapped to the 5′ and 3′ ends or mature tRNAs. (B) Size distribution of all identified tRF reads from four developmental stages in terms of either unique read sequence abundance or total read abundance. (C) The raw abundance of the 5′ tRF and 3′ tRF reads at four developmental stages, as indicated.