Stm1p, a G4 quadruplex and purine motif triplex nucleic acid-binding protein, interacts with ribosomes and subtelomeric Y' DNA in Saccharomyces cerevisiae.

Michael W. Van Dyke,Rodney G. Weilbaecher,Laura D. Nelson,Dakshesh V. Mehta

doi:10.1074/jbc.m401981200

Michael W. Van Dyke, Rodney G. Weilbaecher + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m401981200

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Abstract

The Saccharomyces cerevisiae protein Stm1 was originally identified as a G4 quadruplex and purine motif triplex nucleic acid-binding protein. However, more recent studies have suggested a role for Stm1p in processes ranging from antiapoptosis to telomere maintenance. To better understand the biological role of Stm1p and its potential for G(*)G multiplex binding, we used epitope-tagged protein and immunological methods to identify the subcellular localization and protein and nucleic acid partners of Stm1p in vivo. Indirect immunofluorescence microscopy indicated that Stm1p is primarily a cytoplasmic protein, although a small percentage is also present in the nucleus. Conventional immunoprecipitation found that Stm1p is associated with ribosomal proteins and rRNA. This association was verified by rate zonal separation through sucrose gradients, which showed that Stm1p binds exclusively to mature 80 S ribosomes and polysomes. Chromatin immunoprecipitation experiments found that Stm1p preferentially binds telomere-proximal Y' element DNA sequences. Taken together, our data suggest that Stm1p is primarily a ribosome-associated protein, but one that can also interact with DNA, especially subtelomeric sequences. We discuss the implications of our findings in relation to prior genetic, genomic, and proteomic studies that have identified STM1 and/or Stm1p as well as the possible biological role of Stm1p.

Highlights

The Saccharomyces cerevisiae protein Stm1 was originally identified as a G4 quadruplex and purine motif triplex nucleic acid-binding protein
Stm1p is a moderately abundant (35,000 – 46,800 proteins/ cell) S. cerevisiae protein that has been investigated for a variety of reasons over the last 10 years
Identified by biochemical means as one of the primary G4 quadruplex nucleic acid-binding proteins in yeast whole cell extracts, Stm1p or its corresponding gene has been identified in a number of genetic or biochemical screens

Summary

The abbreviations used are

Purine motif triple helical DNA; ARS, autonomous replicating sequence(s); ChIP, chromatin immunoprecipitation; EMSA, electrophoretic mobility shift assay; G4, Gquartet; G*G, Hoogsteen hydrogen-bonded guanines; HA, influenza hemagglutinin protein epitope; mAb, monoclonal antibody; NTS, nontranscribed sequence; ETS, early transcribed sequence. Ative charge densities due to the presence of multiple phosphate backbones, the simpler designation “G*G multiplex.” Both Pu triplexes and G4 structures can form under physiological conditions and are both extremely stable species once formed. Proteins have been identified in eukaryotes ranging from yeast to humans that and avidly bind Pu triplexes and G4 DNAs and RNAs in vitro (8, 10, 17–28, 30 – 44). Biochemical demonstrations of protein-G*G multiplex interactions in vitro do not necessarily prove that such phenomena occur in vivo, let alone that such species truly exist. To address this important question, we sought to identify those nucleic acids bound by G*G multiplex-binding proteins within living cells. We report that Stm1p is primarily a ribosome-associated protein, it can be found associated with subtelomeric YЈ elements in yeast chromosomes

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION