Single-molecule analysis of the human telomerase RNA.dyskerin interaction and the effect of dyskeratosis congenita mutations.
It has been proposed that human telomerase RNA (hTR) interacts with dyskerin, prior to assembly of the telomerase holoenzyme. The direct interaction of dyskerin and hTR has not been demonstrated and is an experimentally challenging research problem because of difficulties in expressing and purifying dyskerin in quantities that are useful for biophysical analysis. By orthogonally labeling dyskerin and hTR, we have been able to employ single-molecule two-color coincidence detection (TCCD) to observe directly the formation of a dyskerin.hTR complex. By systematic deletion of hTR subdomains, we have gained insights into the RNA sites required for interaction with dyskerin. We then investigated mutated forms of hTR and dyskerin that are associated with dyskeratosis congenita (DC), on the basis of clinical genetics studies, for their effects on the dyskerin.hTR interaction. Dyskerin mutations associated with X-linked DC resulted in significant impairment of the dyskerin.hTR interaction, whereas mutations in hTR associated with autosomal dominant (AD) DC did not affect the interaction. We propose that disruption of the dyskerin.hTR interaction may contribute to X-linked DC.
Ashbridge
Beth
B
University Chemical Laboratories, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.
Orte
Angel
A
Yeoman
Justin A
JA
Kirwan
Michael
M
Vulliamy
Tom
T
Dokal
Inderjeet
I
Klenerman
David
D
Balasubramanian
Shankar
S
D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004262:DNA Restriction Enzymes / Q000737:chemistry; D019871:Dyskeratosis Congenita / Q000235:genetics*; D006801:Humans; D018613:Microscopy, Confocal; D009154:Mutation / Q000235:genetics*; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D012313:RNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012815:Signal Processing, Computer-Assisted; D013050:Spectrometry, Fluorescence; D019098:Telomerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D018797:Cell Cycle Proteins; C112148:DKC1 protein, human; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; C422952:telomerase RNA; D012313:RNA; D019098:Telomerase; D004262:DNA Restriction Enzymes