RESUMO
Many RNAs, including pre-mRNAs and long non-coding RNAs, can be thousands of nucleotides long and undergo complex post-transcriptional processing. Multiple sites of alternative splicing within a single gene exponentially increase the number of possible spliced isoforms, with most human genes currently estimated to express at least ten. To understand the mechanisms underlying these complex isoform expression patterns, methods are needed that faithfully maintain long-range exon connectivity information in individual RNA molecules. In this study, we describe SeqZip, a methodology that uses RNA-templated DNA-DNA ligation to retain and compress connectivity between distant sequences within single RNA molecules. Using this assay, we test proposed coordination between distant sites of alternative exon utilization in mouse Fn1, and we characterize the extraordinary exon diversity of Drosophila melanogaster Dscam1.
Assuntos
Processamento Alternativo , Bioensaio , DNA/genética , Edição de RNA , Precursores de RNA/genética , RNA Longo não Codificante/genética , Animais , Moléculas de Adesão Celular , DNA/química , DNA/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Embrião não Mamífero , Éxons , Fibronectinas/química , Fibronectinas/genética , Fibronectinas/metabolismo , Variação Genética , Humanos , Camundongos , Moléculas de Adesão de Célula Nervosa/química , Moléculas de Adesão de Célula Nervosa/genética , Moléculas de Adesão de Célula Nervosa/metabolismo , Hibridização de Ácido Nucleico , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Precursores de RNA/química , Precursores de RNA/metabolismo , RNA Longo não Codificante/química , RNA Longo não Codificante/metabolismoRESUMO
The SF1 helicase MOV10 is an antiviral factor that is incorporated into human immunodeficiency virus type 1 (HIV-1) virions. We now report that HIV-1 virions also incorporate UPF1, which belongs to the same SF1 helicase subfamily as MOV10 and functions in the nonsense-mediated decay (NMD) pathway. Unlike ectopic MOV10, the overexpression of UPF1 does not impair the infectivity of HIV-1 progeny virions. However, UPF1 becomes a potent inhibitor of HIV-1 progeny virion infectivity when residues required for its helicase activity are mutated. In contrast, equivalent mutations abolish the antiviral activity of MOV10. Importantly, cells depleted of endogenous UPF1, but not of another NMD core component, produce HIV-1 virions of substantially lower specific infectivity. The defect is at the level of reverse transcription, the same stage of the HIV-1 life cycle inhibited by ectopic MOV10. Thus, whereas ectopic MOV10 restricts HIV-1 replication, the related UPF1 helicase functions as a cofactor at an early postentry step.
Assuntos
Interações Hospedeiro-Patógeno , Transativadores/metabolismo , Replicação Viral , HIV-1/fisiologia , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , RNA Helicases , Transativadores/genéticaAssuntos
RNA Interferente Pequeno/metabolismo , Animais , Proteínas Argonautas/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Mapeamento Cromossômico , Camundongos , Família Multigênica , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , RNA Interferente Pequeno/genética , Espermatogênese , Fatores de Transcrição/metabolismoRESUMO
Animal germ cells produce PIWI-interacting RNAs (piRNAs), small silencing RNAs that suppress transposons and enable gamete maturation. Mammalian transposon-silencing piRNAs accumulate early in spermatogenesis, whereas pachytene piRNAs are produced later during postnatal spermatogenesis and account for >95% of all piRNAs in the adult mouse testis. Mutants defective for pachytene piRNA pathway proteins fail to produce mature sperm, but neither the piRNA precursor transcripts nor the trigger for pachytene piRNA production is known. Here, we show that the transcription factor A-MYB initiates pachytene piRNA production. A-MYB drives transcription of both pachytene piRNA precursor RNAs and the mRNAs for core piRNA biogenesis factors including MIWI, the protein through which pachytene piRNAs function. A-MYB regulation of piRNA pathway proteins and piRNA genes creates a coherent feedforward loop that ensures the robust accumulation of pachytene piRNAs. This regulatory circuit, which can be detected in rooster testes, likely predates the divergence of birds and mammals.