Contributions of the TEL-patch amino acid cluster on TPP1 to telomeric DNA synthesis by human telomerase.

Telomere maintenance is a highly coordinated process, and its misregulation is linked to cancer and telomere-shortening syndromes. Recent studies have shown that the TEL-patch--a cluster of amino acids on the surface of the shelterin component TPP1--is necessary for the recruitment of telomerase to the telomere in human cells. However, there has been only basic biochemical analysis of the role of TPP1 in the telomerase recruitment process. Here we develop an in vitro assay to quantitatively measure the contribution of the TEL-patch to telomerase recruitment--binding and extension of the first telomeric repeat. We also demonstrate that the TEL-patch contributes to the translocation step of the telomerase reaction. Finally, our quantitative observations indicate that the TEL-patch stabilizes the association between telomerase and telomeric DNA substrates, providing a molecular explanation for its contributions to telomerase recruitment and action.

Identification of human TERT elements necessary for telomerase recruitment to telomeres.

Human chromosomes terminate in telomeres, repetitive DNA sequences bound by the shelterin complex. Shelterin protects chromosome ends, prevents recognition by the DNA damage machinery, and recruits telomerase. A patch of amino acids, termed the TEL-patch, on the OB-fold domain of the shelterin component TPP1 is essential to recruit telomerase to telomeres. In contrast, the site on telomerase that interacts with the TPP1 OB-fold is not well defined. In this study, we identify separation-of-function mutations in the TEN-domain of human telomerase reverse transcriptase (hTERT) that disrupt the interaction of telomerase with TPP1 in vivo and in vitro but have very little effect on the catalytic activity of telomerase. Suppression of a TEN-domain mutation with a compensatory charge-swap mutation in the TEL-patch indicates that their association is direct. Our findings define the interaction interface required for telomerase recruitment to telomeres, an important step towards developing modulators of this interaction as therapeutics for human disease.

RNA recognition by the DNA end-binding Ku heterodimer.

Most nucleic acid-binding proteins selectively bind either DNA or RNA, but not both nucleic acids. The Saccharomyces cerevisiae Ku heterodimer is unusual in that it has two very different biologically relevant binding modes: (1) Ku is a sequence-nonspecific double-stranded DNA end-binding protein with prominent roles in nonhomologous end-joining and telomeric capping, and (2) Ku associates with a specific stem-loop of TLC1, the RNA subunit of budding yeast telomerase, and is necessary for proper nuclear localization of this ribonucleoprotein enzyme. TLC1 RNA-binding and dsDNA-binding are mutually exclusive, so they may be mediated by the same site on Ku. Although dsDNA binding by Ku is well studied, much less is known about what features of an RNA hairpin enable specific recognition by Ku. To address this question, we localized the Ku-binding site of the TLC1 hairpin with single-nucleotide resolution using phosphorothioate footprinting, used chemical modification to identify an unpredicted motif within the hairpin secondary structure, and carried out mutagenesis of the stem-loop to ascertain the critical elements within the RNA that permit Ku binding. Finally, we provide evidence that the Ku-binding site is present in additional budding yeast telomerase RNAs and discuss the possibility that RNA binding is a conserved function of the Ku heterodimer.

Aptamer-based multiplexed proteomic technology for biomarker discovery.

BACKGROUND: The interrogation of proteomes ("proteomics") in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology and medicine. METHODOLOGY/PRINCIPAL FINDINGS: We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 µL of serum or plasma). Our current assay measures 813 proteins with low limits of detection (1 pM median), 7 logs of overall dynamic range (~100 fM-1 µM), and 5% median coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding signature of DNA aptamer concentrations, which is quantified on a DNA microarray. Our assay takes advantage of the dual nature of aptamers as both folded protein-binding entities with defined shapes and unique nucleotide sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD). We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to rapidly discover unique protein signatures characteristic of various disease states. CONCLUSIONS/SIGNIFICANCE: We describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine.

Specificity and functionality of microRNA inhibitors.

BACKGROUND: Micro(mi)RNAs regulate gene expression through translational attenuation and messenger (m)RNA degradation, and are associated with differentiation, homeostasis and disease. Natural miRNA target recognition is determined primarily by perfect complementarity in a seed region (nucleotide positions 2 to 7) with additional interactions contributing in a sequence- and target-specific manner. Synthetic miRNA target analogs, which are fully complementary, chemically modified oligonucleotides, have been used successfully to inhibit miRNA function. RESULTS: In this paper, we present a first systematic study to evaluate the effect of mismatches in the target site on synthetic inhibitor activity. Panels of miRNA inhibitors containing two-nucleotide mismatches across the target site were tested against three miRNAs (miR-21, miR-22 and miR-122). The results showed that the function of inhibitors vary as mismatch positions in the inhibitors change. CONCLUSIONS: The data indicate that features important for natural miRNA target recognition (such as seed region complementarity) are also important for inhibitor functionality. In addition, base pairing at a second, more 3' region appears to be equally important in determining the efficacy of synthetic inhibitors. Considering the importance of these inhibitor regions and the expression of closely related miRNA sequences will enable researchers to interpret results more accurately in future experiments.

Double-stranded regions are essential design components of potent inhibitors of RISC function.

While microRNAs (miRNAs) are recognized as playing a critical role in regulating eukaryotic gene expression, both the mechanism by which these small, noncoding RNAs function and the genes they target remain elusive. Previous studies have shown that short, single-stranded 2'-O-methyl-modified oligonucleotides that are complementary to mature microRNA sequences can interact with the miRNA-RISC nucleoprotein complex and weakly inhibit miRNA function. Here we report the identification of secondary structural elements that enhance the potency of these molecules. Incorporation of highly structured, double-stranded flanking regions around the reverse complement core significantly increases inhibitor function and allows for multi-miRNA inhibition at subnanomolar concentrations. The improved functionality of these double-stranded miRNA inhibitors may provide insights into the miRNA mechanism by suggesting the possible importance of such structures in or near endogenous miRNA target sites.

Culture-independent analysis of indomethacin-induced alterations in the rat gastrointestinal microbiota.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly prescribed for a variety of inflammatory conditions; however, the benefits of this class of drugs are accompanied by deleterious side effects, most commonly gastric irritation and ulceration. NSAID-induced ulceration is thought to be exacerbated by intestinal microbiota, but previous studies have not identified specific microbes that contribute to these adverse effects. In this study, we conducted a culture-independent analysis of approximately 1,400 bacterial small-subunit rRNA genes associated with the small intestines and mesenteric lymph nodes of rats treated with the NSAID indomethacin. This is the first molecular analysis of the microbiota of the rat small intestine. A comparison of clone libraries and species-specific quantitative PCR results from rats treated with indomethacin and untreated rats revealed that organisms closely related to Enterococcus faecalis were heavily enriched in the small intestine and mesenteric lymph nodes of the treated rats. These data suggest that treatment of NSAID-induced ulceration may be facilitated by addressing the microbiological imbalances.

Structural perspective on the activation of RNAse P RNA by protein.

Ribonucleoprotein particles are central to numerous cellular pathways, but their study in vitro is often complicated by heterogeneity and aggregation. We describe a new technique to characterize these complexes trapped as homogeneous species in a nondenaturing gel. Using this technique, in conjunction with phosphorothioate footprinting analysis, we identify the protein-binding site and RNA folding states of ribonuclease P (RNase P), an RNA-based enzyme that, in vivo, requires a protein cofactor to catalyze the 5' maturation of precursor transfer RNA (pre-tRNA). Our results show that the protein binds to a patch of conserved RNA structure adjacent to the active site and influences the conformation of the RNA near the tRNA-binding site. The data are consistent with a role of the protein in substrate recognition and support a new model of the holoenzyme that is based on a recently solved crystal structure of RNase P RNA.

Protein activation of a ribozyme: the role of bacterial RNase P protein.

Bacterial ribonuclease P (RNase P) belongs to a class of enzymes that utilize both RNAs and proteins to perform essential cellular functions. The bacterial RNase P protein is required to activate bacterial RNase P RNA in vivo, but previous studies have yielded contradictory conclusions regarding its specific functions. Here, we use biochemical and biophysical techniques to examine all of the proposed functions of the protein in both Escherichia coli and Bacillus subtilis RNase P. We demonstrate that the E. coli protein, but not the B. subtilis protein, stabilizes the global structure of RNase P RNA, although both proteins influence holoenzyme dimer formation and precursor tRNA recognition to different extents. By comparing each protein in complex with its cognate and noncognate RNA, we show that differences between the two types of holoenzymes reside primarily in the RNA and not the protein components of each. Our results reconcile previous contradictory conclusions regarding the role of the protein and support a model where the protein activates local RNA structures that manifest multiple holoenzyme properties.

The scrambled actin I gene in Uroleptus pisces.

The micronuclear gene encoding actin I in Uroleptus pisces occurs in two segments. Segment I contains 638 bp divided into six macronuclear destined subsegments, or MDSs, by five internal eliminated segments, or IESs. The MDSs in segment 1 are in the scrambled disorder, 1-2-4-8-6-15, with MDSs 8 and 6 inverted. Segment II contains 2452 bp divided into ten MDSs by nine IESs in the scrambled disorder, 3-5-7-10-13-12-9-14-16-11, with MDSs 12, 9, and 11 inverted. Extensive attempts by polymerase chain reaction to connect the two segments failed. We conclude that the two segments are separated by a very long IES or are in different loci. The pattern of the 16 scrambled MDSs is entirely different from the scrambled pattern observed for the actin I gene in six other stichotrichs. We conclude that the actin I gene became scrambled on two separate occasions during stichotrich evolution: once in the lineage leading to the group of six stichotrichs, which includes, among others, Sterkiella species and Stylonychia lemnae, and once in the lineage leading to Uroleptus pisces. Repeated sequence pairs (pointers) of three to 14 bases at the ends of MDSs appear to be essential for correct splicing of MDSs during macronuclear development. However, the micronuclear actin gene also contains 40 matches of eight or more bases between IESs and MDSs that do not function as pointers. To prevent these ectopic repeats from causing improper processing of the micronuclear gene appears to demand a template of DNA or RNA from the old macronucleus to guide splicing of MDSs in the orthodox order.

Macronuclear molecules encoding actins in spirotrichs.

The nucleotide sequences of 16 newly reported and 8 previously reported actin-encoding macronuclear DNA molecules in spirotrichs have been compared. As described for the eight previously reported molecules, the first 50 bases (noncoding) inside the telomere at both 5' strands in additional actin molecules are purine-rich. This anomalous base composition might serve as a signal to identify macronuclear molecules in micronuclear DNA during development. The 50-base segment upstream of the ATG in the 5' leaders of the actin molecules contains extensive, conserved sequence motifs that are possibly promoter elements. The 3' noncoding trailers contain virtually no conserved sequence motifs. With one exception, the 3' trailers contain a second stop codon (TGA) 36 bases on average downstream of the primary stop codon. Excluding Moneuplotes crassus, amino acid identities in actin I range from 78 to 100%, with variations distributed nonrandomly along the sequence. Phylogenetic trees based on the actin nucleotide sequences of 22 spirotrichs define the evolutionary relationships of their actin-encoding molecules. The actin phylogeny, while well supported by posterior probabilities, does not always coincide with the phylogeny defined in rDNA analyses or classical taxonomic classifications.