Cotranscriptional effect of a premature termination codon revealed by live-cell imaging.
Aberrant mRNAs with premature translation termination codons (PTCs) are recognized and eliminated by the nonsense-mediated mRNA decay (NMD) pathway in eukaryotes. We employed a novel live-cell imaging approach to investigate the kinetics of mRNA synthesis and release at the transcription site of PTC-containing (PTC+) and PTC-free (PTC-) immunoglobulin-μ reporter genes. Fluorescence recovery after photobleaching (FRAP) and photoconversion analyses revealed that PTC+ transcripts are specifically retained at the transcription site. Remarkably, the retained PTC+ transcripts are mainly unspliced, and this RNA retention is dependent upon two important NMD factors, UPF1 and SMG6, since their depletion led to the release of the PTC+ transcripts. Finally, ChIP analysis showed a physical association of UPF1 and SMG6 with both the PTC+ and the PTC- reporter genes in vivo. Collectively, our data support a mechanism for regulation of PTC+ transcripts at the transcription site.
Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Valeria
V
de Turris
Pamela
P
Nicholson
Rodolfo Zamudio
RZ
Orozco
Robert H
RH
Singer
Oliver
O
Mühlemann
D002460:Cell Line; D018389:Codon, Nonsense; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D023061:Gene Order; D020868:Gene Silencing; D006801:Humans; D007148:Immunoglobulin mu-Chains / Q000235:genetics; D017404:In Situ Hybridization, Fluorescence; D059365:Nonsense Mediated mRNA Decay; D020365:RNA Helicases; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism*; D019098:Telomerase / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D004722:Endoribonucleases
D018389:Codon, Nonsense; D007148:Immunoglobulin mu-Chains; D020365:RNA Helicases; D012333:RNA, Messenger; D019098:Telomerase; D015534:Trans-Activators; C512261:SMG6 protein, human; D004722:Endoribonucleases; C101828:UPF1 protein, human