Deletions of singular U1 snRNA gene significantly interfere with transcription and 3'-end mRNA formation.

Small nuclear RNAs (snRNAs) are structural and functional cores of the spliceosome. In metazoan genomes, each snRNA has multiple copies/variants, up to hundreds in mammals. However, the expressions and functions of each copy/variant in one organism have not been systematically studied. Focus on U1 snRNA genes, we investigated all five copies in Drosophila melanogaster using two series of constructed strains. Analyses of transgenic flies that each have a U1 promoter-driven gfp revealed that U1:21D is the major and ubiquitously expressed copy, and the other four copies have specificities in developmental stages and tissues. Mutant strains that each have a precisely deleted copy of U1-gene exhibited various extents of defects in fly morphology or mobility, especially deletion of U1:82Eb. Interestingly, splicing was changed at limited levels in the deletion strains, while large amounts of differentially-expressed genes and alternative polyadenylation events were identified, showing preferences in the down-regulation of genes with 1-2 introns and selection of proximal sites for 3'-end polyadenylation. In vitro assays suggested that Drosophila U1 variants pulled down fewer SmD2 proteins compared to the canonical U1. This study demonstrates that all five U1-genes in Drosophila have physiological functions in development and play regulatory roles in transcription and 3'-end formation.

Comprehensive Identification and Alternative Splicing of Microexons in Drosophila.

Interrupted exons in the pre-mRNA transcripts are ligated together through RNA splicing, which plays a critical role in the regulation of gene expression. Exons with a length ≤ 30 nt are defined as microexons that are unique in identification. However, microexons, especially those shorter than 8 nt, have not been well studied in many organisms due to difficulties in mapping short segments from sequencing reads. Here, we analyzed mRNA-seq data from a variety of Drosophila samples with a newly developed bioinformatic tool, ce-TopHat. In addition to the Flybase annotated, 465 new microexons were identified. Differentially alternatively spliced (AS) microexons were investigated between the Drosophila tissues (head, body, and gonad) and genders. Most of the AS microexons were found in the head and two AS microexons were identified in the sex-determination pathway gene fruitless.

Defective minor spliceosomes induce SMA-associated phenotypes through sensitive intron-containing neural genes in Drosophila.

The minor spliceosome is evolutionarily conserved in higher eukaryotes, but its biological significance remains poorly understood. Here, by precise CRISPR/Cas9-mediated disruption of the U12 and U6atac snRNAs, we report that a defective minor spliceosome is responsible for spinal muscular atrophy (SMA) associated phenotypes in Drosophila. Using a newly developed bioinformatic approach, we identified a large set of minor spliceosome-sensitive splicing events and demonstrate that three sensitive intron-containing neural genes, Pcyt2, Zmynd10, and Fas3, directly contribute to disease development as evidenced by the ability of their cDNAs to rescue the SMA-associated phenotypes in muscle development, neuromuscular junctions, and locomotion. Interestingly, many splice sites in sensitive introns are recognizable by both minor and major spliceosomes, suggesting a new mechanism of splicing regulation through competition between minor and major spliceosomes. These findings reveal a vital contribution of the minor spliceosome to SMA and to regulated splicing in animals.

HITS-CLIP reveals sex-differential RNA binding and alterative splicing regulation of SRm160 in Drosophila.

Serine/arginine (SR)-rich proteins are critical for the regulation of alternative splicing (AS), which generates multiple mRNA isoforms from one gene and provides protein diversity for cell differentiation and tissue development. Genetic evidence suggests that Drosophila genital-specific overexpression of SR-related nuclear matrix protein of 160 kDa (SRm160), an SR protein with a PWI RNA-binding motif, causes defective development only in male flies and results in abnormal male genital structures and abnormal testis. However, the molecular characterization of SRm160 is limited. Using the high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) method in two sex-specific embryonic cell lines, S2 from the male and Kc from the female, we first identified the genome-wide RNA-binding characteristics of SRm160, which preferred binding to the exonic tri-nucleotide repeats GCA and AAC. We then validated this binding through both in vitro gel-shift assay and in vivo splicing of minigenes and found that SRm160 level affects AS of many transcripts. Furthermore, we identified 492 differential binding sites (DBS) of SRm160 varying between the two sex-specific cell lines. Among these DBS-containing genes, splicing factors were highly enriched, including transformer, a key regulator in the sex determination cascade. Analyses of fly mutants demonstrated that the SRm160 level affects AS isoforms of transformer. These findings shed crucial light on SRm160's RNA-binding specificity and regulation of AS in Drosophila sex determination and development.