The p80 homology region of TEP1 is sufficient for its association with the telomerase and vault RNAs, and the vault particle

TEP1 is a protein component of two ribonucleoprotein complexes: vaults and telomerase. The vault-associated small RNA, termed vault RNA (VR), is dependent upon TEP1 for its stable association with vaults, while the association of telomerase RNA with the telomerase complex is independent of TEP1. Both of these small RNAs have been shown to interact with amino acids 1–871 of TEP1 in an indirect yeast three-hybrid assay. To understand the determinants of TEP1–RNA binding, we generated a series of TEP1 deletions and show by yeast three-hybrid assay that the entire Tetrahymena p80 homology region of TEP1 is required for its interaction with both telomerase and VRs. This region is also sufficient to target the protein to the vault particle. Electrophoretic mobility shift assays using the recombinant TEP1 RNA-binding domain (TEP1–RBD) demonstrate that it binds RNA directly, and that telomerase and VRs compete for binding. VR binds weakly to TEP1–RBD in vitro, but mutation of VR sequences predicted to disrupt helices near its central loop enhances binding. Antisense oligonucleotide-directed RNase H digestion of endogenous VR indicates that this region is largely single stranded, suggesting that TEP1 may require access to the VR central loop for efficient binding

Different modes and potencies of translational repression by sequence-specific RNA–protein interaction at the 5′-UTR

To determine whether sequence-specific RNA–protein interaction at the 5′-untranslated region (5′-UTR) can potently repress translation in mammalian cells, a bicistronic translational repression assay was developed to permit direct assessment of RNA–protein interaction and translational repression in transiently transfected living mammalian cells. Changes in cap-dependent yellow fluorescent protein (YFP) and internal ribosome entry sequence (IRES)-dependent cyan fluorescent protein (CFP) translation were monitored by fluorescence microscopy. Selective repression of YFP or coordinate repression of both YFP and CFP translation occurred, indicating two distinct modes by which RNA-binding proteins repress translation through the 5′-UTR. Interestingly, a single-stranded RNA-binding protein from Bacillus subtilis, tryptophan RNA-binding attenuation protein (TRAP), showed potent translational repression, dependent on the level of TRAP expression and position of its cognate binding site within the bicistronic reporter transcript. As the first of its class to be examined in mammalian cells, its potency in repression of translation through the 5′-UTR may be a general feature for this class of single-stranded RNA-binding proteins. Finally, a one-hybrid screen based on translational repression through the 5′-UTR identified linkers supporting full-translational repression as well as a range of partial repression by TRAP within the context of a fusion protein

Native gel electrophoresis of human telomerase distinguishes active complexes with or without dyskerin

Telomeres, the ends of linear chromosomes, safeguard against genome instability. The enzyme responsible for extension of the telomere 3′ terminus is the ribonucleoprotein telomerase. Whereas telomerase activity can be reconstituted in vitro with only the telomerase RNA (hTR) and telomerase reverse transcriptase (TERT), additional components are required in vivo for enzyme assembly, stability and telomere extension activity. One such associated protein, dyskerin, promotes hTR stability in vivo and is the only component to co-purify with active, endogenous human telomerase. We used oligonucleotide-based affinity purification of hTR followed by native gel electrophoresis and in-gel telomerase activity detection to query the composition of telomerase at different purification stringencies. At low salt concentrations (0.1 M NaCl), affinity-purified telomerase was ‘supershifted’ with an anti-dyskerin antibody, however the association with dyskerin was lost after purification at 0.6 M NaCl, despite the retention of telomerase activity and a comparable yield of hTR. The interaction of purified hTR and dyskerin in vitro displayed a similar salt-sensitive interaction. These results demonstrate that endogenous human telomerase, once assembled and active, does not require dyskerin for catalytic activity. Native gel electrophoresis may prove useful in the characterization of telomerase complexes under various physiological conditions

Genetic variation in five genes important in telomere biology and risk for breast cancer

Telomeres, consisting of TTAGGG nucleotide repeats and a protein complex at chromosome ends, are critical for maintaining chromosomal stability. Genomic instability, following telomere crisis, may contribute to breast cancer pathogenesis. Many genes critical in telomere biology have limited nucleotide diversity, thus, single nucleotide polymorphisms (SNPs) in this pathway could contribute to breast cancer risk. In a population-based study of 1995 breast cancer cases and 2296 controls from Poland, 24 SNPs representing common variation in POT1, TEP1, TERF1, TERF2 and TERT were genotyped. We did not identify any significant associations between individual SNPs or haplotypes and breast cancer risk; however, data suggested that three correlated SNPs in TERT (−1381C>T, −244C>T, and Ex2-659G>A) may be associated with reduced risk of breast cancer among individuals with a family history of breast cancer (odds ratios 0.73, 0.66, and 0.57, 95% confidence intervals 0.53–1.00, 0.46–0.95 and 0.39–0.84, respectively). In conclusion, our data do not support substantial overall associations between SNPs in telomere pathway genes and breast cancer risk. Intriguing associations with variants in TERT among women with a family history of breast cancer warrant follow-up in independent studies

CryoAPEX – an electron tomography tool for subcellular localization of membrane proteins

ABSTRACT We describe a method, termed cryoAPEX, which couples chemical fixation and high-pressure freezing of cells with peroxidase tagging (APEX) to allow precise localization of membrane proteins in the context of a well-preserved subcellular membrane architecture. Further, cryoAPEX is compatible with electron tomography. As an example, we apply cryoAPEX to obtain a high-resolution three-dimensional contextual map of the human FIC (filamentation induced by cAMP) protein, HYPE (also known as FICD). HYPE is a single-pass membrane protein that localizes to the endoplasmic reticulum (ER) lumen and regulates the unfolded protein response. Alternate cellular locations for HYPE have been suggested. CryoAPEX analysis shows that, under normal and/or resting conditions, HYPE localizes robustly within the subdomains of the ER and is not detected in the secretory pathway or other organelles. CryoAPEX is broadly applicable for assessing both lumenal and cytosol-facing membrane proteins.</jats:p

The p80 homology region of TEP1 is sufficient for its association with the telomerase and vault RNAs, and the vault particle.

TEP1 is a protein component of two ribonucleoprotein complexes: vaults and telomerase. The vault-associated small RNA, termed vault RNA (VR), is dependent upon TEP1 for its stable association with vaults, while the association of telomerase RNA with the telomerase complex is independent of TEP1. Both of these small RNAs have been shown to interact with amino acids 1-871 of TEP1 in an indirect yeast three-hybrid assay. To understand the determinants of TEP1-RNA binding, we generated a series of TEP1 deletions and show by yeast three-hybrid assay that the entire Tetrahymena p80 homology region of TEP1 is required for its interaction with both telomerase and VRs. This region is also sufficient to target the protein to the vault particle. Electrophoretic mobility shift assays using the recombinant TEP1 RNA-binding domain (TEP1-RBD) demonstrate that it binds RNA directly, and that telomerase and VRs compete for binding. VR binds weakly to TEP1-RBD in vitro, but mutation of VR sequences predicted to disrupt helices near its central loop enhances binding. Antisense oligonucleotide-directed RNase H digestion of endogenous VR indicates that this region is largely single stranded, suggesting that TEP1 may require access to the VR central loop for efficient binding