Interaction of an Arabidopsis RNA-binding Protein with Plant Single-stranded Telomeric DNA Modulates Telomerase Activity

Abstract

Telomeres are the specialized structures at the end of linear chromosomes and terminate with a singlestranded 3 overhang of the G-rich strand. The primary role of telomeres is to protect chromosome ends from recombination and fusion and from being recognized as broken DNA ends. This protective function can be achieved through association with specific telomerebinding proteins. Although proteins that bind singlestranded G-rich overhang regulate telomere length and telomerase activity in mammals and lower eukaryotes, equivalent factors have yet to be identified in plants.Here we have identified proteins capable of interacting with the G-rich single-stranded telomeric repeat from the Arabidopsis extracts by affinity chromatography. Matrix-assisted laser desorption ionization time-offlight mass spectrometry analysis indicates that the isolated protein is a chloroplast RNA-binding protein (and a truncated derivative). The truncated derivative, which we refer to as STEP1 (single-stranded telomerebinding protein 1), binds specifically the singlestranded G-rich plant telomeric DNA sequences but not double-stranded telomeric DNA. Unlike the chloroplastlocalized full-length RNA-binding protein, STEP1 localizes exclusively to the nucleus, suggesting that it plays a role in plant telomere biogenesis. We also demonstrated that the specific binding of STEP1 to single-stranded telomeric DNA inhibits telomerase-mediated telomere extension. The evidence presented here suggests that STEP1 is a telomere-end binding protein that may contribute to telomere length regulation by capping the ends of chromosomes and thereby repressing telomerase activity in plants.

Phylogenetic and Expression Analysis of RNA-binding Proteins with Triple RNA Recognition Motifs in Plants

“…Several genes in the abscisic acid (ABA) signaling pathway have been identified as RBPs (Hugouvieux et al, 2001;Lu and Fedoroff, 2000). RBPs have been shown to interact with single-stranded telomeric DNA and to repress the telomerase activity (Kwon and Chung, 2004). Further, plant RBPs interact with DEAD box RNA helicases and fibrillarin to form a ribonucleoprotein complex involved in ribosomal RNA metabolism (Gendra et al, 2004).…”

Roles of NtGTBP1 in telomere stability

“…10,12 These results suggest potentially conserved roles of the HnRNP family on telomeres. We cloned three HnRNP homologs in tobacco and termed them NtGTBP1 binding to telomeres was correlated to the inhibition of single-strand invasion.…”

“…9,10 We constructed the RNAi-mediated NtGTBP1 knock down transgenic tobacco plants by introducing hair-pin-forming DNA of NtGTBP1 fragments under the control of the CaMV 35S promoter. The NtGTBP1 knock down plants showed severe developmental Figure 1.…”

“…[6][7][8] These results led us to investigate other proteins that potentially bind to single-stranded telomeric ends. Because some reports have found that human heterogeneous nuclear ribonucleoproteins (HnRNP) homologs contain sequence-specific telomere repeat binding activity in higher plants, 9,10 we characterized tobacco NtGTBP1, a homolog of human HnRNPs, by performing in vitro gel retardation assays and phenotypic analyses of RNAi-mediated knockdown transgenic tobacco plants, in which NtGTBP1 was downregulated. 11…”

Screening a cDNA Library for Protein–Protein Interactions Directly in Planta

“…The nuclear localization of a complex comprising CTE and a RRM-containing protein, encoded by At5g10350, suggests a possible telomeric function for this protein. Several previously characterized telomeric binding proteins from plants and other organisms contain a RRM (Petracek et al, 1994;Hirata et al, 2004;Kwon and Chung, 2004;Liu et al, 2009). Their specific binding to telomeric single-stranded DNA is mediated by a tandem pair of RRM located at the C terminus of the protein.…”