Transcriptional, post-transcriptional and chromatin-associated regulation of pri-miRNAs, pre-miRNAs and moRNAs

Abstract

MicroRNAs (miRNAs) play a major role in the post-transcriptional regulation of target genes, especially in development and differentiation. Our understanding about the transcriptional regulation of miRNA genes is limited by inadequate annotation of primary miRNA (pri-miRNA) transcripts. Here, we used CAGE-seq and RNA-seq to provide genome-wide identification of the pri-miRNA core promoter repertoire and its dynamic usage during zebrafish embryogenesis. We assigned pri-miRNA promoters to 152 precursor-miRNAs (pre-miRNAs), the majority of which were supported by promoter associated post-translational histone modifications (H3K4me3, H2A.Z) and RNA polymerase II (RNAPII) occupancy. We validated seven miR-9 pri-miRNAs by in situ hybridization and showed similar expression patterns as mature miR-9. In addition, processing of an alternative intronic promoter of miR-9-5 was validated by 5' RACE PCR. Developmental profiling revealed a subset of pri-miRNAs that are maternally inherited. Moreover, we show that promoter-associated H3K4me3, H2A.Z and RNAPII marks are not only present at pri-miRNA promoters but are also specifically enriched at pre-miRNAs, suggesting chromatin level regulation of pre-miRNAs. Furthermore, we demonstrated that CAGE-seq also detects 3'-end processing of pre-miRNAs on Drosha cleavage site that correlates with miRNA-offset RNAs (moRNAs) production and provides a new tool for detecting Drosha processing events and predicting pre-miRNA processing by a genome-wide assay.

Figure 1.

Mapping of zebrafish miRNA primary transcripts during embryonic development. (A) Schematic representation of the approach used to predict pri-miRNA transcripts (see Materials and Methods). (B) A genome browser view of pri-miRNA transcript of intergenic miR-17–92 cluster. Full-length transcript is reconstructed from RNA-seq and the promoter transcription start site is defined by CAGE-seq. H3K4me3-modified nucleosomes at 5′-ends provides additional evidence for being a true promoter. Maternal and zygotic transcriptome stages are represented by vertical bars in blue and red, respectively. (C) Distribution of pre-miRNAs with annotated pri-miRNAs (red), other pre-miRNAs without annotated pri-miRNAs (green) and miR-430 pre-miRNAs (blue). (D) Distance of pri-miRNA TSSs relative to pre-miRNA. (E) Number of pri-miRNAs TSSs defined by CAGE-seq that are supported by H3K4me3 peaks and RNA-seq/EST transcripts. (F–H) Alignment of promoter-associated histone modifications (H3K4me3, H2A.Z) and RNAPII supports the annotated pri-miRNAs 5′-ends. Y-axis shows the normalized read count in tags per million (tpm).

Figure 2.

Expression pattern revealed by in situ hybridization of mature miR-9 and pri-miRNA transcripts during prim6 stage. (A) Expression pattern of mature miR-9 is mostly restricted to telencephalon. Left panels represent lateral views and right panels represent dorsal views. (B–I) Expression pattern of miR-9 pri-miRNA transcripts is also restricted to telencephalon with differences in the spatiotemporal details among different pri-miRNAs, such as: miR-9-2 (developing retina, white asterisk; Figure 2C), miR-9-4 (posterior brain regions, white arrowhead; Figure 2E) and miR-9-6 (hypothalamus, white arrow; Figure 2H). (J) Amplified region of 5′ RACE PCR product (corresponding to probe-1) of miR-9-5 maps exactly to the alternative intronic CAGE tags. (K) Unique discrete amplification band obtained from 5′ RACE PCR products obtained for miR-9-5 (with intronic CAGE tags). Multiple non-specific bands and/or a smear were obtained for miR-9-4 that did not have alternative intronic CAGE tags.

Figure 3.

Developmental dynamics of pri-miRNAs and their characteristics features. (A) Clustering of pri-miRNAs based on the activity of annotated promoters across 12 developmental stages. The heat map represents the expression level (log₂(tpm)) at indicated stages. Black rectangle indicates maternally inherited pri-miRNAs. (B) Alignment of sequences based on representative transcription start sites of pri-miRNAs reveal an enrichment of canonical initiators and associated TATA-like motifs. (C) Distribution of sharp and broad promoters and their dynamic usage across developmental stages. X-axis indicates 12 developmental stages, same as in (A). (D) Average G+C content of pri-miRNAs promoters. Y-axis indicates the average GC content.

Figure 4.

Promoter-associated histone modifications at pre-miRNAs differ from those at pri-miRNA promoters. (A) A genome browser view of zebrafish mir-9–2 with CAGE tags, H3K4me3 and H2A.Z tracks. H3K4me3 and H2A.Z peaks at pre-miRNA are distinct from pri-miRNA. (B) A genome browser view of human orthologous MIR-9-2 with H3K4me3 and H3K27ac tracks. (C) Alignment of average H3K4me3 (prim6 stage), H2A.Z and RNAPII (both Dome/30%Epiboly stage) marks along the 5′-ends of pre-miRNAs reveal an enriched peak associated with pre-miRNAs. (D) Heat maps showing H3K4me3, H2A.Z and RNAPII signals of each pre-miRNAs. Pre-miRNAs overlapping CGIs are stacked on top and pre-miRNAs not overlapping CGIs are stacked below in the increasing order of pri-miRNA distance.

Figure 5.

CAGE-seq detects post-transcriptional processing of pre-miRNAs on Drosha cleavage sites. (A) Alignment of CAGE tags around pre-miRNAs reveal post-transcriptionally generated CAGE tags are enriched at Drosha cleavage site during zygotic stages. Pre-miRNAs are schematically represented by rectangular boxes. Broken edges towards 3′-end represents variable length. Red and blue arrows indicate the Dicer and Drosha cleavage sites. (B) A genome browser view of miR-125c with CAGE tags and small RNA tracks from prim6 stage. Black horizontal bar represent annotated mature miRNA. Small RNA reads map to mature miRNA arms and flanking regions (blue blocks). (C) Comparison of length of transcription initiation clusters (TCs) of moRNAs and pri-miRNAs. (D and E) Small RNA reads aligned to 5′-ends of Drosha cleavage sites detected by CAGE-seq from (D) maternal (256 cells) and (E) zygotic (Prim6) transcriptome revealed that moRNA production occurs predominantly in zygotic transcription.

Figure 6.

3′ RACE PCR amplification of miR-9-(1/4/5/6). (A–D) Schematic representations of miR-9-(1/4/5/6) loci and designed primers based on downstream sequences (see Supplementary Table S3). The resulting 3′ RACE PCR products are shown in dark blue. Mature miRNAs sequences are highlighted in orange and the remaining sequences of pre-miRNA hairpins in grey. 5′-end of RACE PCR products (blue bars) precisely mapped Drosha cleavage site (red arrow). The agarose gel images on the right show that unique RACE PCR products were obtained.