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1.
Nucleic Acids Res ; 51(12): 6073-6086, 2023 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-37125647

RESUMO

Many prokaryotic operons encode a processive antitermination (P-AT) system. Transcription complexes associated with an antitermination factor can bypass multiple transcription termination signals regardless of their sequences. However, to avoid compromising transcriptional regulation of downstream regions, the terminator at the end of the operon needs to be resistant to antitermination. So far, no studies on the mechanism of resistance to antitermination have been reported. The recently discovered conAn P-AT system is composed of two components that are encoded at the start of many conjugation operons on plasmids of Gram-positive bacteria. Here we report the identification of a conAn-resistant terminator, named TerR, in the conjugation operon of the Bacillus subtilis plasmid pLS20, re-defining the end of the conjugation operon. We investigated the various characteristics of TerR and show that its extraordinary long stem is the determining feature for resistance to antitermination. This is the first P-AT resistance mechanism to be reported.


Assuntos
Células Procarióticas , Regiões Terminadoras Genéticas , Óperon/genética , Plasmídeos/genética , Fatores de Transcrição , Transcrição Gênica , Células Procarióticas/metabolismo
2.
Cells ; 11(22)2022 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-36429068

RESUMO

Pathogenic variants in RPE65 lead to retinal diseases, causing a vision impairment. In this work, we investigated the pathomechanism behind the frequent RPE65 variant, c.11+5G>A. Previous in silico predictions classified this change as a splice variant. Our prediction using novel software's suggested a 124-nt exon elongation containing a premature stop codon. This elongation was validated using midigenes-based approaches. Similar results were observed in patient-derived induced pluripotent stem cells (iPSC) and photoreceptor precursor cells. However, the splicing defect in all cases was detected at low levels and thereby does not fully explain the recessive condition of the resulting disease. Long-read sequencing discarded other rearrangements or variants that could explain the diseases. Subsequently, a more relevant model was employed: iPSC-derived retinal pigment epithelium (RPE) cells. In patient-derived iPSC-RPE cells, the expression of RPE65 was strongly reduced even after inhibiting a nonsense-mediated decay, contradicting the predicted splicing defect. Additional experiments demonstrated a cell-specific gene expression reduction due to the presence of the c.11+5G>A variant. This decrease also leads to the lack of the RPE65 protein, and differences in size and pigmentation between the patient and control iPSC-RPE. Altogether, our data suggest that the c.11+5G>A variant causes a cell-specific defect in the expression of RPE65 rather than the anticipated splicing defect which was predicted in silico.


Assuntos
Células-Tronco Pluripotentes Induzidas , Splicing de RNA , Humanos , Splicing de RNA/genética , Epitélio Pigmentado da Retina/metabolismo , Éxons/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
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