RESUMO
In the absence of extensive transcription control mechanisms the pathogenic parasite Trypanosoma brucei crucially depends on translation regulation to orchestrate gene expression. However, molecular insight into regulating protein biosynthesis is sparse. Here we analyze the small non-coding RNA (ncRNA) interactome of ribosomes in T. brucei during different growth conditions and life stages. Ribosome-associated ncRNAs have recently been recognized as unprecedented regulators of ribosome functions. Our data show that the tRNAThr 3´half is produced during nutrient deprivation and becomes one of the most abundant tRNA-derived RNA fragments (tdRs). tRNAThr halves associate with ribosomes and polysomes and stimulate translation by facilitating mRNA loading during stress recovery once starvation conditions ceased. Blocking or depleting the endogenous tRNAThr halves mitigates this stimulatory effect both in vivo and in vitro. T. brucei and its close relatives lack the well-described mammalian enzymes for tRNA half processing, thus hinting at a unique tdR biogenesis in these parasites.
Assuntos
Biossíntese de Proteínas/genética , RNA Mensageiro/genética , RNA de Transferência/genética , Ribossomos/genética , Trypanosoma brucei brucei/genética , Polirribossomos/genética , Polirribossomos/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Mensageiro/metabolismo , RNA de Protozoário/genética , RNA de Protozoário/metabolismo , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , RNA de Transferência/metabolismo , RNA de Transferência de Treonina/genética , RNA de Transferência de Treonina/metabolismo , Ribossomos/metabolismo , Estresse Fisiológico , Trypanosoma brucei brucei/metabolismoRESUMO
Spinal Muscular Atrophy is due to the loss of SMN1 gene function. The duplicate gene SMN2 produces some, but not enough, SMN protein because most transcripts lack exon 7. Thus, promoting the inclusion of this exon is a therapeutic option. We show that a somatic gene therapy using the gene for a modified U7 RNA which stimulates this splicing has a profound and persistent therapeutic effect on the phenotype of a severe Spinal Muscular Atrophy mouse model. To this end, the U7 gene and vector and the production of pure, highly concentrated self-complementary (sc) adenovirus-associated virus 9 vector particles were optimized. Introduction of the functional vector into motoneurons of newborn Spinal Muscular Atrophy mice by intracerebroventricular injection led to a highly significant, dose-dependent increase in life span and improvement of muscle functions. Besides the central nervous system, the therapeutic U7 RNA was expressed in the heart and liver which may additionally have contributed to the observed therapeutic efficacy. This approach provides an additional therapeutic option for Spinal Muscular Atrophy and could also be adapted to treat other diseases of the central nervous system with regulatory small RNA genes.
Assuntos
Adenoviridae/genética , Terapia Genética/métodos , Atrofia Muscular Espinal/terapia , RNA Nuclear Pequeno/administração & dosagem , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Animais , Modelos Animais de Doenças , Vetores Genéticos/administração & dosagem , Fígado/metabolismo , Camundongos , Camundongos Transgênicos , Atrofia Muscular Espinal/genética , Miocárdio/metabolismo , Splicing de RNA , RNA Nuclear Pequeno/farmacologiaRESUMO
The RNome of a cell is highly diverse and consists besides messenger RNAs (mRNAs), transfer RNAs (tRNAs), and ribosomal RNAs (rRNAs) also of other small and long transcript entities without apparent coding potential. This class of molecules, commonly referred to as non-protein-coding RNAs (ncRNAs), is involved in regulating numerous biological processes and thought to contribute to cellular complexity. Therefore, much effort is put into their identification and further functional characterization. Here we provide a cost-effective and reliable method for cDNA library construction of small RNAs in the size range of 20-500 residues. The effectiveness of the described method is demonstrated by the analysis of ribosome-associated small RNAs in the eukaryotic model organism Trypanosoma brucei.
