The predicted secondary structure of the TLC1 RNA of S. cerevisiae reveals a distinct folding pattern featuring well-separated but conserved functional elements. The predicted structure now allows for a detailed and rationally designed study to the structure-function relationships within the telomerase RNP-complex in a genetically tractable system.

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 ⌬rnt1 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.

The RNA component of budding yeast telomerase (Tlc1) occurs in two forms, a non-polyadenylated form found in functional telomerase and a rare polyadenylated version with unknown function. Previous work suggested that the functional Tlc1 polyA− RNA is processed from the polyA+ form, but the mechanisms regulating its transcription termination and 3′-end formation remained unclear. Here we examined transcription termination of Tlc1 RNA in the sequences 3′ of the TLC1 gene and relate it to telomere maintenance. Strikingly, disruption of all probable or cryptic polyadenylation signals near the 3′-end blocked the accumulation of the previously reported polyA+ RNA without affecting the level, function or specific 3′ nucleotide of the mature polyA− form. A genetic approach analysing TLC1 3′-end sequences revealed that transcription terminates upstream of the polyadenylation sites. Furthermore, the results also demonstrate that the function of this Tlc1 terminator depends on the Nrd1/Nab3 transcription termination pathway. The data thus show that transcription termination of the budding yeast telomerase RNA occurs as that of snRNAs and Tlc1 functions in telomere maintenance are not strictly dependent on a polyadenylated precursor, even if the polyA+ form can serve as intermediate in a redundant termination/maturation pathway.

In bakers' yeast, in vivo telomerase activity requires a ribonucleoprotein (RNP) complex with at least four associated proteins (Est2p, Est1p, Est3p, and Cdc13p) and one RNA species (Tlc1). The function of telomerase in maintaining chromosome ends, called telomeres, is tightly regulated and linked to the cell cycle. However, the mechanisms that regulate the expression of individual components of telomerase are poorly understood. Here we report that yeast RNase III (Rnt1p), a double-stranded RNA-specific endoribonuclease, regulates the expression of telomerase subunits and is required for maintaining normal telomere length. Deletion or inactivation of RNT1 induced the expression of Est1, Est2, Est3, and Tlc1 RNAs and increased telomerase activity, leading to elongation of telomeric repeat tracts. In silico analysis of the different RNAs coding for the telomerase subunits revealed a canonical Rnt1p cleavage site near the 3 end of Est1 mRNA. This predicted structure was cleaved by Rnt1p and its disruption abolished cleavage in vitro. Mutation of the Rnt1p cleavage signal in vivo impaired the cell cycle-dependent degradation of Est1 mRNA without affecting its steady-state level. These results reveal a new mechanism that influences telomeres length by controlling the expression of the telomerase subunits.The ends of eukaryotic chromosomes are capped with special structures made of tandem DNA repeats and associated proteins, called telomeres. These structures protect chromosomes from end-to-end fusion, recombination, and nucleolytic degradation (1, 2). However, because the conventional DNA replication machinery cannot fully duplicate the ends of linear chromosomes, telomeric DNA will shorten with each round of replication, leading to a replicative senescence (3). To solve this end replication problem, most eukaryotes use the activity of an enzyme called telomerase to ensure the maintenance of telomeric DNA (4). Telomerase is a ribonucleoprotein reverse transcriptase that can extend the 3Ј end of chromosomes using its RNA subunit as a template for the addition of telomeric repeats.In vitro, telomerase activity requires a reverse transcriptase (Tert, Est2p in yeast) and an RNA template (Terc, Tlc1 in yeast)

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 snoRNAassociated 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.In eukaryotes, the 25S/28S, 18S, and 5.8S rRNAs are produced by RNA polymerase I as single RNA transcripts that are processed, modified, and assembled into ribosomes within the nucleolus (41). In Saccharomyces cerevisiae, trimming of the nascent pre-rRNA 3Ј end and RNA modifications produce the first stable rRNA precursor (50). The 35S pre-rRNA is subjected to a series of further cleavages that remove external transcribed spacers 1 and 2 and internal transcribed spacers 1 and 2 to produce mature rRNA. Changes in the sequence of cleavage or modification sites within the pre-rRNA transcript usually alter the cleavage pattern at distal sites, indicating an interdependent processing pathway (3,5,36). However, the mechanistic basis for this coordination remains undefined.At least six endonucleolytic cleavages are required for the production of mature rRNA in yeast cells, and two endonucleases are known to be involved in rRNA maturation (50). The first is RNase MRP, a ribonucleoprotein endonuclease related to the tRNA processing enzyme RNase P (43). MRP cleaves at site A3, one of two redundant sites that lead to the formation of mature 18S rRNA (46). The second endonuclease is the double-stranded RNA (dsRNA)-specific RNase (Rnt1p), which is the orthologue of the bacterial pre-rRNA processing enzyme RNase III (2, 40). Rnt1p performs the earliest cleavage event at the 3Ј end of the pre-rRNA transcript immediately after transcription (2). Rnt1p is also required for the processing of several snRNAs (1,11,47,49) and snoRNAs, including members of both C/D and H/ACA snoRNA families (12). Rnt1p accurately cleaves most of the snoRNA substrates in vitro in the absence of other factors, with the exception of U18, which requires the presence of the C/D small nucleolar ribonucleoprotein (snoRNP) Nop1p (17). Rnt1p is required for normal growth at 30°C and is essential for growth at 37°C (31). Deletion of the enzyme inhibits the cleavage of the 25S prerRNA and delays cleavage at site A0 upstream of the 18S rRNA (2, 26). Like MRP, Rnt1p cleaves model substrates at sites identical t...

How thrombocytopenia relates to bleeding in 22q11 deletion syndrome (22q11DS) is not clear. Bleeding severity, platelet count and volume, and GPIBB were examined in patients with 22q11DS. Macrothrombocytopenia and bleeding typified imperfectly overlapping subsets of 22q11DS patients. GPIBB hemizygosity does not cause macrothrombocytopenia or bleeding in patients with 22q11DS. Summary Background and objectivesMacrothrombocytopenia and bleeding are frequently associated with 22q11 deletion syndrome (22q11DS). GPIBB, which encodes the glycoprotein (GP) Ibβ subunit of GPIb–IX–V, is commonly deleted in patients with 22q11DS. Absence of functional GPIb–IX–V causes Bernard–Soulier syndrome, which is a severe bleeding disorder characterized by macrothrombocytopenia. Patients with 22q11DS are often obligate hemizygotes for GPIBB, and those with only a pathogenically disrupted copy of GPIBB present with Bernard–Soulier syndrome. The objective of this study was to determine how GPIBB hemizygosity and sequence variation relate to macrothrombocytopenia and bleeding in patients with 22q11DS who do not have Bernard‐Soulier syndrome. Patients/methodsWe thoroughly characterized bleeding severity, mean platelet volume, platelet count and GPIBB copy number and sequence in patients with 22q11DS. Results and conclusionsMacrothrombocytopenia and mild bleeding were observed in incompletely overlapping subsets of patients, and GPIBB copy number and sequence variation did not correlate with either macrothrombocytopenia or bleeding in patients with 22q11DS. These findings indicate that GPIBB hemizygosity does not result in either macrothrombocytopenia or bleeding in these patients. Alternative genetic causes of macrothrombocytopenia, potential causes of acquired thrombocytopenia and bleeding and ways in which platelet size, platelet count and GPIBB sequence information can be used to aid in the diagnosis and management of patients with 22q11DS are discussed.

Telomerase is a specialized ribonucleoprotein that adds repeated DNA sequences to the ends of eukaryotic chromosomes to preserve genome integrity. Some secondary structure features of the telomerase RNA are very well conserved, and it serves as a central scaffold for the binding of associated proteins. The Saccharomyces cerevisiae telomerase RNA, TLC1, is found in very low copy number in the cell and is the limiting component of the known telomerase holoenzyme constituents. The reasons for this low abundance are unclear, but given that the RNA is very stable, transcriptional control mechanisms must be extremely important. Here we define the sequences forming the TLC1 promoter and identify the elements required for its low expression level, including enhancer and repressor elements. Within an enhancer element, we found consensus sites for Mbp1/Swi4 association, and chromatin immunoprecipitation (ChIP) assays confirmed the binding of Mbp1 and Swi4 to these sites of the TLC1 promoter. Furthermore, the enhancer element conferred cell cycle-dependent regulation to a reporter gene, and mutations in the Mbp1/Swi4 binding sites affected the levels of telomerase RNA and telomere length. Finally, ChIP experiments using a TLC1 RNA-binding protein as target showed cell cycle-dependent transcription of the TLC1 gene. These results indicate that the budding yeast TLC1 RNA is transcribed in a cell cycle-dependent fashion late in G1 and may be part of the S phase-regulated group of genes involved in DNA replication.

The last few years have witnessed the appreciation of dsRNA as a regulator of gene expression, a potential antiviral agent, and a tumor suppressor. However, in spite of these clear effects on the cell function, the mechanism that controls dsRNA maturation and stability remains unknown. Recently, the discovery of eukaryotic orthologues of the bacterial dsRNA specific ribonuclease III (RNase III) suggested a central role for these enzymes in the regulation of dsRNA and eukaryotic RNA metabolism in general. This article reviews the structure-function features of the eukaryotic RNase III family and their roles in dsRNA metabolism with an emphasis on the yeast RNase III. Yeast RNase III is involved in the maturation of the majority of snRNAs, snoRNAs, and rRNA. In addition, perturbation of the expression level of yeast RNase III alters meiosis and causes sterility. These basic functions of the yeast RNase III appear to be widely conserved which makes it a good model to understand the importance of eukaryotic dsRNA metabolism. The RNase III Family: A Confusion or Ordered Evolution? Author: NA Khan (2015) www.caister.com/acanthamoeba2 • Microarrays: Current Technology, Innovations and Applications Edited by: Z He (2014) www.caister.com/microarrays2 • Metagenomics of the Microbial Nitrogen Cycle: Theory, Methods and Applications Edited by: D Marco (2014) www.caister.com/n2Caister Academic Press is a leading academic publisher of advanced texts in microbiology, molecular biology and medical research.