The role of the 5'-3' exoribonuclease XRNA in transcriptome-wide mRNA degradation.

The steady-state level of each mRNA in a cell is a balance between synthesis and degradation. Here, we use high-throughput RNA sequencing (RNASeq) to determine the relationship between mRNA degradation and mRNA abundance on a transcriptome-wide scale. The model organism used was the bloodstream form of Trypanosoma brucei, a protist that lacks regulation of RNA polymerase II initiation. The mRNA half-lives varied over two orders of magnitude, with a median half-life of 13 min for total (rRNA-depleted) mRNA. Data for poly(A)+ RNA yielded shorter half-lives than for total RNA, indicating that removal of the poly(A) tail was usually the first step in degradation. Depletion of the major 5'-3' exoribonuclease, XRNA, resulted in the stabilization of most mRNAs with half-lives under 30 min. Thus, on a transcriptome-wide scale, degradation of most mRNAs is initiated by deadenylation. Trypanosome mRNA levels are strongly influenced by gene copy number and mRNA half-life: Very abundant mRNAs that are required throughout the life-cycle may be encoded by multicopy genes and have intermediate-to-long half-lives; those encoding ribosomal proteins, with one to two gene copies, are exceptionally stable. Developmentally regulated transcripts with a lower abundance in the bloodstream forms than the procyclic forms had half-lives around the median, whereas those with a higher abundance in the bloodstream forms than the procyclic forms, such as those encoding glycolytic enzymes, had longer half-lives.

The role of deadenylation in the degradation of unstable mRNAs in trypanosomes.

Removal of the poly(A) tail is the first step in the degradation of many eukaryotic mRNAs. In metazoans and yeast, the Ccr4/Caf1/Not complex has the predominant deadenylase activity, while the Pan2/Pan3 complex may trim poly(A) tails to the correct size, or initiate deadenylation. In trypanosomes, turnover of several constitutively-expressed or long-lived mRNAs is not affected by depletion of the 5'-3' exoribonuclease XRNA, but is almost completely inhibited by depletion of the deadenylase CAF1. In contrast, two highly unstable mRNAs, encoding EP procyclin and a phosphoglycerate kinase, PGKB, accumulate when XRNA levels are reduced. We here show that degradation of EP mRNA was partially inhibited after CAF1 depletion. RNAi-targeting trypanosome PAN2 had a mild effect on global deadenylation, and on degradation of a few mRNAs including EP. By amplifying and sequencing degradation intermediates, we demonstrated that a reduction in XRNA had no effect on degradation of a stable mRNA encoding a ribosomal protein, but caused accumulation of EP mRNA fragments that had lost substantial portions of the 5' and 3' ends. The results support a model in which trypanosome mRNAs can be degraded by at least two different, partially independent, cytoplasmic degradation pathways attacking both ends of the mRNA.

The global spread of rotavirus G10 strains: Detection in Ghanaian children hospitalized with diarrhea.

From October 2003 through September 2004, a total of 289 stool samples were collected from children <5 years of age who had severe diarrhea at admission to or when visiting the emergency department at the Navrongo War Memorial Hospital in rural Ghana during a study on rotavirus disease burden. Rotavirus antigen was detected in 115 stool samples (39.8%) tested for rotavirus. Four rotavirus-positive samples were found to bear G10P[6] specificity by reverse-transcription polymerase chain reaction, polymerase chain reaction-enzyme-linked immunosorbent assay, and oligonucleotide microarray hybridization. Two of these strains further exhibited serotype G10 specificity by neutralization and subgroup II specificity by enzyme immunoassay and possessed long electropheretic patterns by polyacrylamide gel electrophoresis. Their VP7 genes shared a much closer nucleotide identity with other African human G10 strains (>97%) than with human G10 strain from Asia or South America (<86%) or animal strains (<85%). The VP8* genes of the Ghanaian G10 strains exhibited >94% identity to that of human P[6] virus strains and belonged to the P[6] lineage 1a. The deduced VP7 amino acid sequence showed that the Ghanaian strains were more closely related to human G10 strains than to animal G10 strains. The possession of the typical human subgroup II specificity and the P[6] specificity (frequently found in Ghana and the rest of Africa) and the marked similarity in the VP7 antigenic sites suggest that these G10 strains may have evolved through genetic reassortment between bovine and human strains.

Proteins and lipids of glycosomal membranes from Leishmania tarentolae and Trypanosoma brucei.

In kinetoplastid protists, several metabolic pathways, including glycolysis and purine salvage, are located in glycosomes, which are microbodies that are evolutionarily related to peroxisomes. With the exception of some potential transporters for fatty acids, and one member of the mitochondrial carrier protein family, proteins that transport metabolites across the glycosomal membrane have yet to be identified. We show here that the phosphatidylcholine species composition of Trypanosoma brucei glycosomal membranes resembles that of other cellular membranes, which means that glycosomal membranes are expected to be impermeable to small hydrophilic molecules unless transport is facilitated by specialized membrane proteins. Further, we identified 464 proteins in a glycosomal membrane preparation from Leishmania tarentolae. The proteins included approximately 40 glycosomal matrix proteins, and homologues of peroxisomal membrane proteins - PEX11, GIM5A and GIM5B; PXMP4, PEX2 and PEX16 - as well as the transporters GAT1 and GAT3. There were 27 other proteins that could not be unambiguously assigned to other compartments, and that had predicted trans-membrane domains. However, no clear candidates for transport of the major substrates and intermediates of energy metabolism were found. We suggest that, instead, these metabolites are transported via pores formed by the known glycosomal membrane proteins.

Depletion of trypanosome CTR9 leads to gene expression defects.

The Paf complex of Opisthokonts and plants contains at least five subunits: Paf1, Cdc73, Rtf1, Ctr9, and Leo1. Mutations in, or loss of Paf complex subunits have been shown to cause defects in histone modification, mRNA polyadenylation, and transcription by RNA polymerase I and RNA polymerase II. We here investigated trypanosome CTR9, which is essential for trypanosome survival. The results of tandem affinity purification suggested that trypanosome CTR9 associates with homologues of Leo1 and Cdc73; genes encoding homologues of Rtf1 and Paf1 were not found. RNAi targeting CTR9 resulted in at least ten-fold decreases in 131 essential mRNAs: they included several that are required for gene expression and its control, such as those encoding subunits of RNA polymerases, exoribonucleases that target mRNA, RNA helicases and RNA-binding proteins. Simultaneously, some genes from regions subject to chromatin silencing were derepressed, possibly as a secondary effect of the loss of two proteins that are required for silencing, ISWI and NLP1.

A search for Trypanosoma brucei rhodesiense diagnostic antigens by proteomic screening and targeted cloning.

BACKGROUND: The only available diagnostic method for East African trypanosomiasis is light microscopy of blood samples. A simple immunodiagnostic would greatly aid trypanosomiasis control. METHODOLOGY AND PRINCIPAL FINDINGS: To find trypanosome proteins that are specifically recognised by sera from human sleeping sickness patients, we have screened the Trypanosoma brucei brucei proteome by Western blotting. Using cytosolic, cytoskeletal and glycosomal fractions, we found that the vast majority of abundant trypanosome proteins is not specifically recognised by patient sera. We identified phosphoglycerate kinase (PGKC), heat shock protein (HSP70), and histones H2B and H3 as possible candidate diagnostic antigens. These proteins, plus paraflagellar rod protein 1, rhodesain (a cysteine protease), and an extracellular fragment of the Trypanosoma brucei nucleoside transporter TbNT10, were expressed in E. coli and tested for reactivity with patient and control sera. Only TbHSP70 was preferentially recognized by patient sera, but the sensitivity and specificity were insufficient for use of TbHSP70 alone as a diagnostic. Immunoprecipitation using a native protein extract revealed no specifically reacting proteins. CONCLUSIONS: No abundant T. brucei soluble, glycosomal or cytoskeletal protein is likely to be useful in diagnosis. To find useful diagnostic antigens it will therefore be necessary to use more sophisticated proteomic methods, or to test a very large panel of candidate proteins.

DRBD1 is the Trypanosoma brucei homologue of the spliceosome-associated protein 49.

The 5'-ends of all Kinetoplastid mRNAs consist of a short sequence added by trans splicing. In contrast to cis splicing in mammals, trans splicing in trypanosomes does not involve sequence-specific recognition of the branch point by the U2 snRNP. In mammalian cells and yeast, U2 snRNP is associated with the multimeric factor SF3b, which contains p14, SF3b10, SF3b14b, SAP49, SAP130, SAP145 and SAP155. The interaction between Trypanosoma cruzi p14 and SAP155 has already been characterised using the yeast 2-hybrid system. We here identify the Trypanosoma brucei RRM-protein DRBD1 as a homologue of SAP49. TAP-tagged DRBD1 co-purified with homologues of p14, SAP130, SAP145 and SAP155. Tagged DRBD1 was found in the nucleus; RNAi targeting DRBD1 inhibited trypanosome growth and caused a very mild splicing defect.