RESUMO
U3 snoRNA and the associated Rrp9/U3-55K protein are essential for 18S rRNA production by the SSU-processome complex. U3 and Rrp9 are required for early pre-rRNA cleavages at sites A0, A1 and A2, but the mechanism remains unclear. Substitution of Arg 289 in Rrp9 to Ala (R289A) specifically reduced cleavage at sites A1 and A2. Surprisingly, R289 is located on the surface of the Rrp9 ß-propeller structure opposite to U3 snoRNA. To understand this, we first characterized the protein-protein interaction network of Rrp9 within the SSU-processome. This identified a direct interaction between the Rrp9 ß-propeller domain and Rrp36, the strength of which was reduced by the R289A substitution, implicating this interaction in the observed processing phenotype. The Rrp9 R289A mutation also showed strong synergistic negative interactions with mutations in U3 that destabilize the U3/pre-rRNA base-pair interactions or reduce the length of their linking segments. We propose that the Rrp9 ß-propeller and U3/pre-rRNA binding cooperate in the structure or stability of the SSU-processome. Additionally, our analysis of U3 variants gave insights into the function of individual segments of the 5'-terminal 72-nt sequence of U3. We interpret these data in the light of recently reported SSU-processome structures.
Assuntos
Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Ribossômico 18S/metabolismo , RNA Nucleolar Pequeno/química , Ribonucleoproteínas Nucleolares Pequenas/química , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Mutação , Proteínas Nucleares/metabolismo , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , RNA Nucleolar Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The revolution of miRNA discovery, in the early 2000s, shed a new light in the exciting field of small non-coding RNAs. Since then, and owing to outstanding breakthroughs in RNomic techniques, novel small non-coding RNA families have been regularly discovered, e.g., piRNAs, tiRNAs, and many others.In this review, we provide a very succinct historical and functional overview on most prominent small non-coding RNA families.
Assuntos
Genética/história , Modelos Genéticos , Pequeno RNA não Traduzido/genética , História do Século XX , História do Século XXI , Pequeno RNA não Traduzido/históriaRESUMO
Alcoholic precipitation is a critical step to recover RNA of high purity. This chapter describes the principles of alcoholic precipitation as well as a standard, basic protocol with key advices to observe, but numerous variations on the theme are discussed. Indeed, several important parameters, such as the choice of salt, alcohol, or carrier, have to be considered to improve the efficiency of precipitation and the yield of RNA recovery.
Assuntos
2-Propanol/química , Precipitação Química/efeitos dos fármacos , Técnicas de Química Analítica/métodos , Etanol/química , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/isolamento & purificaçãoRESUMO
Small nucleolar RNAs or snoRNAs, principally implicated in post-transcriptional chemical modification of other RNAs, were among the first non-coding RNA identified, together with ribosomal and transfer RNA. Lately, snoRNA have been involved in various unexpected functions, which renewed researcher's interest for these molecules. SnoRNA processing into smaller functional RNA species (sdRNA for snoRNA-derived RNA) or into miRNA (sno-miR), snoRNA mediated regulation of messenger RNA alternative splicing or snoRNA links to human disorders, including cancers, are some of the topics developed in this review.
