RESUMO
The non-coding RNA 7SK is the scaffold for a small nuclear ribonucleoprotein (7SKsnRNP) which regulates the function of the positive transcription elongation factor P-TEFb in the control of RNA polymerase II elongation in metazoans. The La-related protein LARP7 is a component of the 7SKsnRNP required for stability and function of the RNA. To address the function of LARP7 we determined the crystal structure of its La module, which binds a stretch of uridines at the 3'-end of 7SK. The structure shows that the penultimate uridine is tethered by the two domains, the La-motif and the RNA-recognition motif (RRM1), and reveals that the RRM1 is significantly smaller and more exposed than in the La protein. Sequence analysis suggests that this impacts interaction with 7SK. Binding assays, footprinting and small-angle scattering experiments show that a second RRM domain located at the C-terminus binds the apical loop of the 3' hairpin of 7SK, while the N-terminal domains bind at its foot. Our results suggest that LARP7 uses both its N- and C-terminal domains to stabilize 7SK in a closed structure, which forms by joining conserved sequences at the 5'-end with the foot of the 3' hairpin and has thus functional implications.
Assuntos
RNA Nuclear Pequeno/química , Ribonucleoproteínas/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Estabilidade de RNA , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Ribonucleosídeo Difosfato Redutase/química , Ribonucleosídeo Difosfato Redutase/metabolismo , Espalhamento a Baixo Ângulo , Homologia de Sequência de Aminoácidos , Eletricidade Estática , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismo , Uridina/química , Difração de Raios XRESUMO
[This corrects the article DOI: 10.1093/narcan/zcaa036.].
RESUMO
Recent epitranscriptomics studies unravelled that ribosomal RNA (rRNA) 2'O-methylation is an additional layer of gene expression regulation highlighting the ribosome as a novel actor of translation control. However, this major finding lies on evidences coming mainly, if not exclusively, from cellular models. Using the innovative next-generation RiboMeth-seq technology, we established the first rRNA 2'O-methylation landscape in 195 primary human breast tumours. We uncovered the existence of compulsory/stable sites, which show limited inter-patient variability in their 2'O-methylation level, which map on functionally important sites of the human ribosome structure and which are surrounded by variable sites found from the second nucleotide layers. Our data demonstrate that some positions within the rRNA molecules can tolerate absence of 2'O-methylation in tumoral and healthy tissues. We also reveal that rRNA 2'O-methylation exhibits intra- and inter-patient variability in breast tumours. Its level is indeed differentially associated with breast cancer subtype and tumour grade. Altogether, our rRNA 2'O-methylation profiling of a large-scale human sample collection provides the first compelling evidence that ribosome variability occurs in humans and suggests that rRNA 2'O-methylation might represent a relevant element of tumour biology useful in clinic. This novel variability at molecular level offers an additional layer to capture the cancer heterogeneity and associates with specific features of tumour biology thus offering a novel targetable molecular signature in cancer.