Maximizing confidence in a negative result: Quantitative sample adequacy control.

Quantitative PCR (qPCR) is a leading screening tool, permitting rapid detection of pathogens and the maintenance of effective infection control programs. Unfortunately, qPCR assays frequently do not incorporate Sample Adequacy Control (SAC). A SAC controls for the quantity, quality and adequacy of the specimen. Without SAC, the confidence in a negative result remains questionable and the efficacy of screening is compromised. Ultimately, the exclusion of SAC from qPCR may result in false negative results. One should consider SAC to be an integral critical type of laboratory control; addressing diverse analytical problems, such as sample adequacy, sample processing and assay inhibition. Following distribution of cycle threshold values (Cq) of Influenza A positive results and Cq values of SAC, obtained from nasopharyngeal swabs, we showed that the confidence in a negative result cannot be guaranteed in the presence of a weak positive SAC signal (late Cq values). Herein, we explain why widespread inclusion of sample adequacy control in routine screening is blocked. A protocol and methods for SAC threshold establishment are offered.

RNase III-mediated silencing of a glucose-dependent repressor in yeast.

Members of the RNase III family are found in all species examined with the exception of archaebacteria, where the functions of RNase III are carried out by the bulge-helix-bulge nuclease (BHB). In bacteria, RNase III contributes to the processing of many noncoding RNAs and directly cleaves several cellular and phage mRNAs. In eukaryotes, orthologs of RNase III participate in the biogenesis of many miRNAs and siRNAs, and this biogenesis initiates the degradation or translational repression of several mRNAs. However, the capacity of eukaryotic RNase IIIs to regulate gene expression by directly cleaving within the coding sequence of mRNAs remains speculative. Here we show that Rnt1p, a member of the RNase III family, selectively inhibits gene expression in baker's yeast by directly cleaving a stem-loop structure within the mRNA coding sequence. Analysis of mRNA expression upon the deletion of Rnt1p revealed an upregulation of the glucose-dependent repressor Mig2p. Mig2p mRNA became more stable upon the deletion of Rnt1p and resisted glucose-dependent degradation. In vitro, Rnt1p cleaved Mig2p mRNA and a silent mutation that disrupts Rnt1p signals blocked Mig2p mRNA degradation. These observations reveal a new RNase III-dependent mechanism of eukaryotic mRNA degradation.

Identification of the genetic determinants responsible for retinal degeneration in families of Mexican descent.

PURPOSE: To investigate the clinical characteristics and genetic basis of inherited retinal degeneration (IRD) in six unrelated pedigrees from Mexico. METHODS: A complete ophthalmic evaluation including measurement of visual acuities, Goldman kinetic or Humphrey dynamic perimetry, Amsler test, fundus photography, and color vision testing was performed. Family history and blood samples were collected from available family members. DNA from members of two pedigrees was examined for known mutations using the APEX ARRP genotyping microarray and one pedigree using the APEX LCA genotyping microarray. The remaining three pedigrees were analyzed using a custom-designed targeted capture array covering the exons of 233 known retinal degeneration genes. Sequencing was performed on Illumina HiSeq. Reads were mapped against hg19, and variants were annotated using GATK and filtered by exomeSuite. Segregation and ethnicity-matched control sample analyses were performed by dideoxy sequencing. RESULTS: Six pedigrees with IRD were analyzed. Nine rare or novel, potentially pathogenic variants segregating with the phenotype were detected in IMPDH1, USH2A, RPE65, ABCA4, and FAM161A genes. Among these, six were known mutations while the remaining three changes in USH2A, RPE65, and FAM161A genes have not been previously reported to be associated with IRD. Analysis of 100 ethnicity-matched controls did not detect the presence of these three novel variants indicating, these are rare variants in the Mexican population. CONCLUSIONS: Screening patients diagnosed with IRD from Mexico identified six known mutations and three rare or novel potentially damaging variants in IMPDH1, USH2A, RPE65, ABCA4, and FAM161A genes that segregated with disease.

A physical interaction between Gar1p and Rnt1pi is required for the nuclear import of H/ACA small nucleolar RNA-associated proteins.

During rRNA biogenesis, multiple RNA and protein substrates are modified and assembled through the coordinated activity of many factors. In Saccharomyces cerevisiae, the double-stranded RNA nuclease Rnt1p and the H/ACA snoRNA pseudouridylase complex participate in the transformation of the nascent pre-rRNA transcript into 35S pre-rRNA. Here we demonstrate the binding of a component of the H/ACA complex (Gar1p) to Rnt1p in vivo and in vitro in the absence of other factors. In vitro, Rnt1p binding to Gar1p is mutually exclusive of its RNA binding and cleavage activities. Mutations in Rnt1p that disrupt Gar1p binding do not inhibit RNA cleavage in vitro but slow RNA processing, prevent nucleolar localization of H/ACA snoRNA-associated proteins, and reduce pre-rRNA pseudouridylation in vivo. These results demonstrate colocalization of various components of the rRNA maturation complex and suggest a mechanism that links rRNA pseudouridylation and cleavage factors.

Cell cycle-dependent nuclear localization of yeast RNase III is required for efficient cell division.

Members of the double-stranded RNA-specific ribonuclease III (RNase III) family were shown to affect cell division and chromosome segregation, presumably through an RNA interference-dependent mechanism. Here, we show that in Saccharomyces cerevisiae, where the RNA interference machinery is not conserved, an orthologue of RNase III (Rnt1p) is required for progression of the cell cycle and nuclear division. The deletion of Rnt1p delayed cells in both G1 and G2/M phases of the cell cycle. Nuclear division and positioning at the bud neck were also impaired in Deltarnt1 cells. The cell cycle defects were restored by the expression of catalytically inactive Rnt1p, indicating that RNA cleavage is not essential for cell cycle progression. Rnt1p was found to exit from the nucleolus to the nucleoplasm in the G2/M phase, and perturbation of its localization pattern delayed the progression of cell division. A single mutation in the Rnt1p N-terminal domain prevented its accumulation in the nucleoplasm and slowed exit from mitosis without any detectable effects on RNA processing. Together, the data reveal a new role for a class II RNase III in the cell cycle and suggest that at least some members of the RNase III family possess catalysis-independent functions.

Novel germline mutations in the calreticulin gene: implications for the diagnosis of myeloproliferative neoplasms.

Mutations in the calreticulin (CALR) gene are found in the majority of Janus kinase 2-negative myeloproliferative neoplasms MPN and, thus far, have exclusively been reported as acquired, somatic mutations. We assessed the mutational status of exon 9 of the CALR gene in 2000 blood samples submitted to our centre and identified 12 subjects (0.6%) harbouring distinctive CALR mutations, all with an allelic frequency of 50% and all involving indels occurring as multiples of 3 bp. Buccal cell samples obtained from these patients confirmed the germline nature of the mutations. Importantly, these germline mutations were not diagnostic of MPN. We thus report for the first time the identification and confirmation of germline mutations in CALR distinct from those somatic mutations that define classical MPN. The finding of a non-standard CALR mutation with an allelic frequency of 50% should raise suspicion of the possibility of a germline CALR mutation and these cases investigated further.

Molecular requirements for duplex recognition and cleavage by eukaryotic RNase III: discovery of an RNA-dependent DNA cleavage activity of yeast Rnt1p.

Members of the double-stranded RNA (dsRNA) specific RNase III family are known to use a conserved dsRNA-binding domain (dsRBD) to distinguish RNA A-form helices from DNA B-form ones, however, the basis of this selectivity and its effect on cleavage specificity remain unknown. Here, we directly examine the molecular requirements for dsRNA recognition and cleavage by the budding yeast RNase III (Rnt1p), and compare it to both bacterial RNase III and fission yeast RNase III (Pac1). We synthesized substrates with either chemically modified nucleotides near the cleavage sites, or with different DNA/RNA combinations, and investigated their binding and cleavage by Rnt1p. Substitution for the ribonucleotide vicinal to the scissile phosphodiester linkage with 2'-deoxy-2'-fluoro-beta-d-ribose (2' F-RNA), a deoxyribonucleotide, or a 2'-O-methylribonucleotide permitted cleavage by Rnt1p, while the introduction of a 2', 5'-phosphodiester linkage permitted binding, but not cleavage. This indicates that the position of the phosphodiester link with respect to the nuclease domain, and not the 2'-OH group, is critical for cleavage by Rnt1p. Surprisingly, Rnt1p bound to a DNA helix capped with an NGNN tetraribonucleotide loop indicating that the binding of at least one member of the RNase III family is not restricted to RNA. The results also suggest that the dsRBD may accommodate B-form DNA duplexes. Interestingly, Rnt1p, but not Pac1 nor bacterial RNase III, cleaved the DNA strand of a DNA/RNA hybrid, indicating that A-form RNA helix is not essential for cleavage by Rnt1p. In contrast, RNA/DNA hybrids bound to, but were not cleaved by Rnt1p, underscoring the critical role for the nucleotide located at 3' end of the tetraloop and suggesting an asymmetrical mode of substrate recognition. In cell extracts, the native enzyme effectively cleaved the DNA/RNA hybrid, indicating much broader Rnt1p substrate specificity than previously thought. The discovery of this novel RNA-dependent deoxyribonuclease activity has potential implications in devising new antiviral strategies that target actively transcribed DNA.

Sequence dependence of substrate recognition and cleavage by yeast RNase III.

Yeast Rnt1p is a member of the double-stranded RNA (dsRNA) specific RNase III family of endoribonucleases involved in RNA processing and RNA interference (RNAi). Unlike other RNase III enzymes, which recognize a variety of RNA duplexes, Rnt1p cleaves specifically RNA stems capped with the conserved AGNN tetraloop. This unusual substrate specificity challenges the established dogma for substrate selection by RNase III and questions the dsRNA contribution to recognition by Rnt1p. Here we show that the dsRNA sequence adjacent to the tetraloop regulates Rnt1p cleavage by interfering with RNA binding. In context, sequences surrounding the cleavage site directly influence the cleavage efficiency. Introduction of sequences that stabilize the RNA helix enhanced binding while reducing the turnover rate indicating that, unlike the tetraloop, Rnt1p binding to the dsRNA helix may become rate-limiting. These results suggest that Rnt1p activity is strictly regulated by a combination of primary and tertiary structural elements allowing a substrate-specific binding and cleavage efficiency.

Short RNA guides cleavage by eukaryotic RNase III.

In eukaryotes, short RNAs guide a variety of enzymatic activities that range from RNA editing to translation repression. It is hypothesized that pre-existing proteins evolved to bind and use guide RNA during evolution. However, the capacity of modern proteins to adopt new RNA guides has never been demonstrated. Here we show that Rnt1p, the yeast orthologue of the bacterial dsRNA-specific RNase III, can bind short RNA transcripts and use them as guides for sequence-specific cleavage. Target cleavage occurred at a constant distance from the Rnt1p binding site, leaving the guide RNA intact for subsequent cleavage. Our results indicate that RNase III may trigger sequence-specific RNA degradation independent of the RNAi machinery, and they open the road for a new generation of precise RNA silencing tools that do not trigger a dsRNA-mediated immune response.

Evaluation of the RNA determinants for bacterial and yeast RNase III binding and cleavage.

Bacterial double-stranded RNA-specific RNase III recognizes the A-form of an RNA helix with little sequence specificity. In contrast, baker yeast RNase III (Rnt1p) selectively recognizes NGNN tetraloops even when they are attached to a B-form DNA helix. To comprehend the general mechanism of RNase III substrate recognition, we mapped the Rnt1p binding signal and directly compared its substrate specificity to that of both Escherichia coli RNase III and fission yeast RNase III (PacI). Rnt1p bound but did not cleave long RNA duplexes without NGNN tetraloops, whereas RNase III indiscriminately cleaved all RNA duplexes. PacI cleaved RNA duplexes with some preferences for NGNN-capped RNA stems under physiological conditions. Hydroxyl radical footprints indicate that Rnt1p specifically interacts with the NGNN tetraloop and its surrounding nucleotides. In contrast, Rnt1p interaction with GAAA-capped hairpins was weak and largely unspecific. Certain duality of substrate recognition was exhibited by PacI but not by bacterial RNase III. E. coli RNase III recognized RNA duplexes longer than 11 bp with little specificity, and no specific features were required for cleavage. On the other hand, PacI cleaved long, but not short, RNA duplexes with little sequence specificity. PacI cleavage of RNA stems shorter than 27 bp was dependent on the presence of an UU-UC internal loop two nucleotides upstream of the cleavage site. These observations suggest that yeast RNase IIIs have two recognition mechanisms, one that uses specific structural features and another that recognizes general features of the A-form RNA helix.