A siRNA-based screen for genes involved in chromosome end protection

PLoS One. 2011;6(6):e21407. doi: 10.1371/journal.pone.0021407. Epub 2011 Jun 23.

Abstract

Telomeres are nucleoprotein complexes which protect the ends of linear chromosomes from detection as DNA damage and provide a sequence buffer against replication-associated shortening. In mammals, telomeres consist of repetitive DNA sequence (TTAGGG) and associated proteins. The telomeric core complex is called shelterin and is comprised of the proteins TRF1, TRF2, POT1, TIN2, TPP1 and RAP1. Excessive telomere shortening or de-protection of telomeres through the loss of shelterin subunits allows the detection of telomeres as DNA damage, which can be visualized as DNA damage protein foci at chromosome ends called TIF (Telomere Dysfunction-Induced Foci). We sought to exploit the TIF phenotype as marker for telomere dysfunction to identify novel genes involved in telomere protection by siRNA-mediated knock-down of a set of 386 candidates. Here we report the establishment, specificity and feasibility of such a screen and the results of the genes tested. Only one of the candidate genes showed a unique TIF phenotype comparable to the suppression of the main shelterin components TRF2 or TRF1 and that gene was identified as a TRF1-like pseudogene. We also identified a weak TIF phenotype for SKIIP (SNW1), a splicing factor and transcriptional co-activator. However, the knock-down of SKIIP also induced a general, not telomere-specific DNA damage response, which complicates conclusions about a telomeric role. In summary, this report is a technical demonstration of the feasibility of a cell-based screen for telomere deprotection with the potential of scaling it to a high-throughput approach.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA Damage / genetics
  • Gene Knockdown Techniques
  • HeLa Cells
  • Humans
  • Phenotype
  • RNA, Small Interfering / genetics*
  • Shelterin Complex
  • Telomere / genetics*
  • Telomere-Binding Proteins

Substances

  • ACD protein, human
  • RNA, Small Interfering
  • Shelterin Complex
  • Telomere-Binding Proteins