Cryptococcus neoformans: the yeast that likes it hot

The ability of fungi to grow well at mammalian body temperatures is a fundamental characteristic of invasive human fungal pathogens. Cryptococcus neoformans, with its genetics, molecular biology, robust animal models and clinical importance, has become a premier fungal model system for molecular fungal pathogenesis studies. There has been over a half century of study into how C. neoformans grows at high temperatures. However, recently the understanding of high-temperature growth at a molecular level has dramatically accelerated. Many strategies have been used to identify genes and over a dozen genes have already been identified to be necessary for high-temperature growth. It is likely that there are many more to discover. It is clear that, as further studies understand how this encapsulated yeast is able to withstand the stresses of high temperature at a genetic and molecular basis, we will also know more about how it and other fungal pathogens have evolved into well-established human pathogens.     

A novel U2 and U11/U12 snRNP protein that associates with the pre-mRNA branch site

Previous UV cross-linking studies demonstrated that, upon integration of the U2 snRNP into the spliceosome, a 14 kDa protein (p14) interacts directly with the branch adenosine, the nucleophile for the first transesterification step of splicing. We have identified the cDNA encoding this protein by microsequencing a 14 kDa protein isolated from U2-type spliceosomes. This protein contains an RNA recognition motif and is highly conserved across species. Antibodies raised against this cDNA-encoded protein precipitated the 14 kDa protein cross-linked to the branch adenosine, confirming the identity of the p14 cDNA. A combination of immunoblotting, protein microsequencing and immunoprecipitation revealed that p14 is a component of both 17S U2 and 18S U11/U12 snRNPs, suggesting that it contributes to the interaction of these snRNPs with the branch sites of U2- and U12-type pre-mRNAs, respectively. p14 was also shown to be a subunit of the heteromeric splicing factor SF3b and to interact directly with SF3b155. Immuno precipitations indicated that p14 is present in U12-type spliceosomes, consistent with the idea that branch point selection is similar in the major and minor spliceosomes.     

A 5'----3' exoribonuclease of Saccharomyces cerevisiae: size and novel substrate specificity

The purification scheme for a 5'----3' exoribonuclease of Saccharomyces cerevisiae has been modified to facilitate purification of larger amounts of enzyme and further extended to yield highly purified enzyme by use of poly(A)-agarose chromatography. As determined by either sodium dodecyl sulfate-polyacrylamide gel electrophoresis or physical characterization, the enzyme has a molecular weight of about 160,000. Further studies of its substrate specificity show that poly(C) and poly(U) preparations require 5' phosphorylation for activity and that poly(A) with a 5'-triphosphate end group is hydrolyzed at only 12% of the rate of poly(A) with a 5'-monophosphate end group. DNA is not hydrolyzed, but synthetic polydeoxyribonucleotides are strong competitive inhibitors of the hydrolysis of noncomplementary ribopolymers. Poly(A).poly(U) and poly(A).poly(dT) are hydrolyzed at 60 and 50%, respectively, of the rate of poly(A) at 37 degrees C. The RNase H activity of the enzyme can also be demonstrated using an RNA X M13 DNA hybrid as a substrate. When poly(dT).poly(dA) with a 5'-terminal poly(A) segment on the poly(dA) is used as a substrate, the enzyme hydrolyzes the poly(A) "tail," removing the last ribonucleotide, but does not hydrolyze the poly(dA).     

Protein kinase CK2: an enzyme that likes to be different

Protein kinase CK2 (formerly known as casein kinase 2) was among the first protein kinases to be identified and characterized. Surprisingly, in spite of intense efforts, the regulation and cellular functions of CK2 remain obscure. However, recent data on its molecular structure, its signal-mediated intracellular dynamic localization and its unexpected function in cell survival have raised new interest in this enzyme. These studies reveal unique features of CK2 and highlight its importance in the transduction of survival signals.     

A novel member of the RNase D exoribonuclease family functions in mitochondrial guide RNA metabolism in Trypanosoma brucei

RNA turnover and RNA editing are essential for regulation of mitochondrial gene expression in Trypanosoma brucei. RNA turnover is controlled in part by RNA 3' adenylation and uridylation status, with trans-acting factors also impacting RNA homeostasis. However, little is known about the mitochondrial degradation machinery or its regulation in T. brucei. We have identified a mitochondrial exoribonuclease, TbRND, whose expression is highly up-regulated in the insect proliferative stage of the parasite. TbRND shares sequence similarity with RNase D family enzymes but differs from all reported members of this family in possessing a CCHC zinc finger domain. In vitro, TbRND exhibits 3' to 5' exoribonuclease activity, with specificity toward uridine homopolymers, including the 3' oligo(U) tails of guide RNAs (gRNAs) that provide the sequence information for RNA editing. Several lines of evidence generated from RNAi-mediated knockdown and overexpression cell lines indicate that TbRND functions in gRNA metabolism in vivo. First, TbRND depletion results in gRNA tails extended by 2-3 nucleotides on average. Second, overexpression of wild type but not catalytically inactive TbRND results in a substantial decrease in the total gRNA population and a consequent inhibition of RNA editing. The observed effects on the gRNA population are specific as rRNAs, which are also 3'-uridylated, are unaffected by TbRND depletion or overexpression. Finally, we show that gRNA binding proteins co-purify with TbRND. In summary, TbRND is a novel 3' to 5' exoribonuclease that appears to have evolved a function highly specific to the mitochondrion of trypanosomes.     

ISG20L2, a novel vertebrate nucleolar exoribonuclease involved in ribosome biogenesis

Proteomics analyses of human nucleoli provided molecular bases for an understanding of the multiple functions fulfilled by these nuclear domains. However, the biological roles of about 100 of the identified proteins are unpredictable. The present study describes the functional characterization of one of these proteins, ISG20L2. We demonstrate that ISG20L2 is a 3' to 5' exoribonuclease involved in ribosome biogenesis at the level of 5.8 S rRNA maturation, more specifically in the processing of the 12 S precursor rRNA. The use of truncated forms of ISG20L2 demonstrated that its N-terminal half promotes the nucleolar localization and suggested that its C-terminal half bears the exoribonuclease activity. Identification of the binding partners of ISG20L2 confirmed its involvement in the biogenesis of the large ribosomal subunit. These results strongly support the notion that, in human, as it was demonstrated in yeast, 5.8 S rRNA maturation requires several proteins in addition to the exosome complex. Furthermore this observation greatly sustains the idea that the extremely conserved need for correctly processed rRNAs in vertebrates and yeast is achieved by close but different mechanisms.     

XMAP215: a tip tracker that really moves

XMAP215 is a microtubule plus-end binding protein implicated in modulating microtubule dynamics. In this issue, Brouhard et al. (2008) propose a new mechanism to explain how XMAP215 promotes microtubule growth. They report that XMAP215 moves with the growing microtubule plus ends where it catalyzes the addition of tubulin subunits.     

Can supernaturals really heal? A view of science that shows how they might

This article does not have an abstract     

Identification of novel snRNA-specific Sm proteins that bind selectively to U2 and U4 snRNAs in Trypanosoma brucei

In eukaryotes the seven Sm core proteins bind to U1, U2, U4, and U5 snRNAs. In Trypanosoma brucei, Sm proteins have been implicated in binding both spliced leader (SL) and U snRNAs. In this study, we examined the function of these Sm proteins using RNAi silencing and protein purification. RNAi silencing of each of the seven Sm genes resulted in accumulation of SL RNA as well as reduction of several U snRNAs. Interestingly, U2 was unaffected by the loss of SmB, and both U2 and U4 snRNAs were unaffected by the loss of SmD3, suggesting that these snRNAs are not bound by the heptameric Sm complex that binds to U1, U5, and SL RNA. RNAi silencing and protein purification showed that U2 and U4 snRNAs were bound by a unique set of Sm proteins that we termed SSm (specific spliceosomal Sm proteins). This is the first study that identifies specific core Sm proteins that bind only to a subset of spliceosomal snRNAs.     

A novel thermophilic glycosynthase that effects branching glycosylation

A novel thermophilic glycosynthase that effects branching glycosylation has been obtained by mutation of the nucleophile in the active site of the glycosidase from Sulfolobus solfataricus. Two methods for the use of this mutant are reported.     

A novel Drosophila serpin that inhibits serine proteases

Serpins define a large protein family in which most members function as serine protease inhibitors. Here we report the results of a search for serpins in Drosophila melanogaster that are potentially required for oogenesis or embryogenesis. We cloned and sequenced ovarian cDNAs that encode six distinct proteins having extensive sequence similarity to mammalian serpins, including residues important in the serpin inhibition mechanism. One of these new serpins in recombinant form inactivates, and complexes with, trypsin-like proteases in vitro. To our knowledge, these results represent the first evidence for a serpin in Drosophila that functions as a serine protease inhibitor.     

A novel glycoprotein that binds to hyaluronic acid

Hyaluronic acid binding protein (HBP) has been purified to homogeneity from normal rat brain by using Hyaluronate-Sepharose affinity chromatography. It appears as a single band in non-dissociating gel electrophoresis. The molecular weight of native protein, as determined by gel filtration is found to be 68,000 daltons, and has a single subunit of molecular weight approximately 13,500 as determined under denaturing conditions in polyacrylamide gel electrophoresis, indicating that this protein is apparently composed of five identical subunits. Amino acid analysis shows the purified HBP to be rich in glycine and glutamic acid content, and is distinct from fibronectin, link proteins, and gelatin binding proteins which are known to bind to hyaluronic acid. This protein is further characterised as sialic acid containing glycoprotein.