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Overlapping activities of ELAV/Hu family RNA binding proteins specify the extended neuronal 3' UTR landscape in Drosophila [L1 CNS RNA-seq]

ABSTRACT: The goals of this study are to perform quantitative comparison of whole transcriptome of Drosophila melanogaster 1st instar larvae central neural system (L1CNS) from three genotypes: Canton-S, elav5 single mutant, and elav5/fne double mutant; and the global 3'-end features of Drosophila melanogaster 1st instar larvae central neural system from three genotypes: Canton-S, elav5 single mutant. Total RNAs were extracted from dissected L1CNS of Drosophila melanogaster Canton-S, elav5 single mutant, and elav5/fne double mutant; total RNA-seq libraries were prepared using Illumina TruSeq Stranded Total RNA Sample Prep kit with replicates for each genotype; final cDNA libraries were sequenced on Illumina HiSeq-1000 sequencer with PE-100 mode. Total RNA-seq data were mapped to the corresponding UCSC genome assemblies: Drosophila melanogaster (dm6), HISAT2 aligner was used for the alignment with default parameters. We found that L1CNS from Canton-S expresses neural specific 3' UTR extensions like head, while in L1CNS from elav5 single mutant, global neural specific 3' UTR extensions are not significantly lost, but in elav5/fne double mutant L1CNS, there is a dramatic loss of neural specific 3' UTR extensions. We reported for the first time demonstration of trans-acting factors that globally maintain this very distinctive tissue-specific APA landscape.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE155532 | GEO | 2020/10/01

REPOSITORIES: GEO

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Overlapping activities of ELAV/Hu family RNA binding proteins specify the extended neuronal 3' UTR landscape in Drosophila [L1 CNS 3'seq]

Project description:The goal of this study is to perform quantitative comparison of transcriptome-wide 3'-end features of Drosophila melanogaster 1st instar larvae central neural system (L1CNS) from three genotypes: Canton-S, elav5 single mutant, and elav5/fne double mutant; Total RNAs were extracted from dissected L1CNS of Drosophila melanogaster Canton-S, elav5 single mutant, and elav5/fne double mutant; 3'-end sequencing libraries were prepared using Lexogen QuantSeq 3′ mRNA-Seq Library Prep Kit REV for Illumina with replicates for each genotype; final cDNA libraries were sequenced on Illumina HiSeq-1000 sequencer with SE-50 mode. 3'-end sequencing data were mapped onto the corresponding UCSC genome assemblies: Drosophila melanogaster (dm6) and 3′ end clusters were derived and quantified within a 25 bp window. We found that L1CNS from Canton-S expresses neural specific 3' UTR extensions like head, while in L1CNS from elav5 single mutant, global neural specific 3' UTR extensions are not significantly lost, but in elav5/fne double mutant L1CNS, there is a dramatic loss of neural specific 3' UTR extensions. We reported for the first time demonstration of trans-acting factors that globally maintain this very distinctive tissue-specific APA landscape.

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Overlapping activities of ELAV/Hu family RNA binding proteins specify the extended neuronal 3' UTR landscape in Drosophila [S2 3-seq]

Project description:The goals of this study are to establish a cell line based analysis platform to unbiasedly evaluate the capabilities of Elav/Hu family RNA binding proteins in regulating neural specific 3' UTR extension; and perform quantitative analysis of global 3' UTR extension regulated by individual Elav/Hu family RNA binding protein. We reported that in non-neural tissue context such as S2R+ cells, with the overexpression of wildtype Elav, Rbp9 and Fne, we observed global 3' UTR extensions for hundreds of genes, whose 3' UTR extension corespond with those detected in head; also, RNA rocognition motif mutant versions of Elav, Rbp9 and Fne loss the ability to regulate the generation of neural specific 3' UTR extension. We reported here for the first time, we show quantitative comparison between Elav/Hu family RNA binding proteins in regulating neural specific 3' UTR landscape and imply the overlapping activities of Elav/Hu family RNA binding proteins specifying neural 3' UTR landscape in Drosophila.

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Project description:ELAV/Hu factors are conserved RNA binding proteins that play diverse roles in mRNA processing and regulation. The founding member, Drosophila Elav, was recognized as a vital neural factor 35 years ago. Nevertheless, still little is known about its impacts on the transcriptome, and potential functional overlap with its paralogs. Building on our recent findings that neural-specific lengthened 3' UTR isoforms are co-determined by ELAV/Hu factors, we address their impacts on splicing. While only a few splicing targets of Drosophila are known, we find that ectopic expression of the three family members (Elav, Fne and Rbp9) induces overlapping changes to hundreds of cassette exon and dozens of alternative last exon (ALE) splicing events. Reciprocally, double mutants of elav/fne, but not elav alone, have opposite effects on both classes of regulated mRNA processing events in the larval CNS. While manipulation of Drosophila ELAV/Hu factors induces both exon skipping and inclusion, motif analysis indicates their major direct effects are to suppress cassette exon usage. Moreover, the roles of ELAV/HU factors in global promotion of distal ALE splicing are mechanistically linked to terminal 3' UTR extensions in neurons, since both involve local suppression of proximal polyadenylation signals via ELAV/Hu binding sites downstream of cleavage sites. The phenotypic impact of combined ELAV/Hu activities in neural mRNA processing is overt, since fne loss strongly enhances neuronal differentiation phenotypes in elav mutants. Finally, we provide evidence for conservation in mammalian neurons, which undergo broad programs of distal ALE and APA lengthening, linked to ELAV/Hu motifs downstream of regulated polyadenylation sites. Overall, ELAV/Hu proteins orchestrate multiple conserved programs of neuronal mRNA processing by suppressing alternative exons and polyadenylation sites.

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