ABSTRACT
The number of annotated long noncoding RNAs (lncRNAs) continues to grow; however, their functional characterization in model organisms has been hampered by the lack of reliable genetic inactivation strategies. While partial or full deletions of lncRNA loci disrupt lncRNA expression, they do not permit the formal association of a phenotype with the encoded transcript. Here, we examined several alternative strategies for generating lncRNA null alleles in zebrafish and found that they often resulted in unpredicted changes to lncRNA expression. Removal of the transcription start sites (TSSs) of lncRNA genes resulted in hypomorphic mutants, due to the usage of either constitutive or tissue-specific alternative TSSs. Deletions of short, highly conserved lncRNA regions can also lead to overexpression of truncated transcripts. In contrast, knock-in of a polyadenylation signal enabled complete inactivation of malat1, the most abundant vertebrate lncRNA. In summary, lncRNA null alleles require extensive in vivo validation, and we propose insertion of transcription termination sequences as the most reliable approach to generate lncRNA-deficient zebrafish.
Subject(s)
Gene Silencing , RNA, Long Noncoding/genetics , Zebrafish/genetics , Animals , CRISPR-Cas Systems , Gene Expression Regulation , Gene Knock-In Techniques , Organ Specificity , Sequence Deletion , Transcription Initiation SiteABSTRACT
Super-enhancers (SEs) are key transcriptional drivers of cellular, developmental, and disease states in mammals, yet the conservational and regulatory features of these enhancer elements in nonmammalian vertebrates are unknown. To define SEs in zebrafish and enable sequence and functional comparisons to mouse and human SEs, we used genome-wide histone H3 lysine 27 acetylation (H3K27ac) occupancy as a primary SE delineator. Our study determined the set of SEs in pluripotent state cells and adult zebrafish tissues and revealed both similarities and differences between zebrafish and mammalian SEs. Although the total number of SEs was proportional to the genome size, the genomic distribution of zebrafish SEs differed from that of the mammalian SEs. Despite the evolutionary distance separating zebrafish and mammals and the low overall SE sequence conservation, â¼42% of zebrafish SEs were located in close proximity to orthologs that also were associated with SEs in mouse and human. Compared to their nonassociated counterparts, higher sequence conservation was revealed for those SEs that have maintained orthologous gene associations. Functional dissection of two of these SEs identified conserved sequence elements and tissue-specific expression patterns, while chromatin accessibility analyses predicted transcription factors governing the function of pluripotent state zebrafish SEs. Our zebrafish annotations and comparative studies show the extent of SE usage and their conservation across vertebrates, permitting future gene regulatory studies in several tissues.
Subject(s)
Chromatin/genetics , Conserved Sequence/genetics , Enhancer Elements, Genetic , Zebrafish/genetics , Acetylation , Animals , Embryonic Development/genetics , Gene Expression Regulation, Developmental , Genomics , Histones/genetics , Humans , Mice , Transcription Factors/geneticsABSTRACT
Spermatogenesis is associated with major and unique changes to chromosomes and chromatin. Here, we sought to understand the impact of these changes on spermatogenic transcriptomes. We show that long terminal repeats (LTRs) of specific mouse endogenous retroviruses (ERVs) drive the expression of many long non-coding transcripts (lncRNA). This process occurs post-mitotically predominantly in spermatocytes and round spermatids. We demonstrate that this transposon-driven lncRNA expression is a conserved feature of vertebrate spermatogenesis. We propose that transposon promoters are a mechanism by which the genome can explore novel transcriptional substrates, increasing evolutionary plasticity and allowing for the genesis of novel coding and non-coding genes. Accordingly, we show that a small fraction of these novel ERV-driven transcripts encode short open reading frames that produce detectable peptides. Finally, we find that distinct ERV elements from the same subfamilies act as differentially activated promoters in a tissue-specific context. In summary, we demonstrate that LTRs can act as tissue-specific promoters and contribute to post-mitotic spermatogenic transcriptome diversity.
Subject(s)
DNA Transposable Elements , Evolution, Molecular , Spermatogenesis , Transcription, Genetic , Animals , Endogenous Retroviruses/genetics , Genomics , Male , Mice , Open Reading Frames , Promoter Regions, Genetic , RNA, Long Noncoding/genetics , Spermatocytes/physiology , Terminal Repeat Sequences , TranscriptomeABSTRACT
microRNAs (miRNAs) repress target transcripts through partial complementarity. By contrast, highly complementary miRNA-binding sites within viral and artificially engineered transcripts induce miRNA degradation in vitro and in cell lines. Here, we show that a genome-encoded transcript harboring a near-perfect and deeply conserved miRNA-binding site for miR-29 controls zebrafish and mouse behavior. This transcript originated in basal vertebrates as a long noncoding RNA (lncRNA) and evolved to the protein-coding gene NREP in mammals, where the miR-29-binding site is located within the 3' UTR. We show that the near-perfect miRNA site selectively triggers miR-29b destabilization through 3' trimming and restricts its spatial expression in the cerebellum. Genetic disruption of the miR-29 site within mouse Nrep results in ectopic expression of cerebellar miR-29b and impaired coordination and motor learning. Thus, we demonstrate an endogenous target-RNA-directed miRNA degradation event and its requirement for animal behavior.