Fibrillarin-associated box C/D small nucleolar RNAs in Trypanosoma brucei. Sequence conservation and implications for 2'-O-ribose methylation of rRNA

We report the identification of 17 box C/D fibrillarin-associated small nucleolar RNAs (snoRNAs) from the ancient eukaryote, Trypanosoma brucei. To systematically isolate and characterize these snoRNAs, the T. brucei cDNA for the box C/D snoRNA common protein, fibrillarin, was cloned and polyclonal antibodies to the recombinant fibrillarin protein were generated in rabbits. Immunoprecipitations from T. brucei extracts with the anti-fibrillarin antibodies indicated that this trypanosomatid has at least 30 fibrillarin-associated snoRNAs. We have sequenced seventeen of them and designated them TBR for T. brucei RNA 1-17. All of them bear conserved box C, D, C', and D' elements, a hallmark of fibrillarin-associated snoRNAs in eukaryotes. Fourteen of them are novel T. brucei snoRNAs. Fifteen bear potential guide regions to mature rRNAs suggesting that they are involved in 2'-O-ribose methylation. Indeed, eight ribose methylations have been mapped in the rRNA at sites predicted by the snoRNA sequences. Comparative genomics indicates that six of the seventeen are the first trypanosome homologs of known yeast and vertebrate methylation guide snoRNAs. Our results indicate that T. brucei has many fibrillarin-associated box C/D snoRNAs with roles in 2'-O-ribose methylation of rRNA and that the mechanism for targeting the nucleotide to be methylated at the fifth nucleotide upstream of box D or D' originated in early eukaryotes.     

Depletion of anaphase-promoting complex or cyclosome (APC/C) subunit homolog APC1 or CDC27 of Trypanosoma brucei arrests the procyclic form in metaphase but the bloodstream form in anaphase

The anaphase-promoting complex or cyclosome (APC/C) is a multiprotein subunit E3 ubiquitin ligase complex that controls segregation of chromosomes and exit from mitosis in eukaryotes. It triggers elimination of key cell cycle regulators such as securin and mitotic cyclins during mitosis by polyubiquitinating them for proteasome degradation. Seven core subunit homologs of APC/C (APC1, APC2, APC11, CDC16, CDC23, CDC27, and DOC1) were identified in the Trypanosoma brucei genome data base. Expression of six of them was individually ablated by RNA interference in both the procyclic and bloodstream forms of T. brucei. Only the CDC27- and APC1-depleted cells were enriched in the G2/M phase with inhibited growth. Further studies indicated that T. brucei APC1 and CDC27 failed to complement the corresponding deletion mutants of budding yeast. However, their depletion from procyclic-form T. brucei enriched cells with two kinetoplasts and an enlarged nucleus possessing short metaphase-like mitotic spindles, suggesting that APC1 and CDC27 may play essential roles in promoting anaphase in the procyclic form. Their depletion from the bloodstream form, however, enriched cells with two kinetoplasts and two nuclei connected through a microtubule bundle, suggesting a late anaphase arrest. This is the first time functional APC/C subunit homologs were identified in T. brucei. The apparent differential activities of this putative APC/C in two distinct developmental stages suggest an unusual function. The apparent lack of functional involvement of some of the other individual structural subunit homologs of APC/C may indicate the structural uniqueness of T. brucei APC/C.     

The roles of intersubunit interactions in exosome stability

In eukaryotes, at least 10 proteins associate in a 3'-5' exonuclease complex, the exosome, which is involved in the processing of many RNA species. A recent model for the exosome placed six RNase PH-related components in a hexameric ring core structure, with three S1 domain proteins associated with the ring surface. So far, however, this model lacks experimental support. Using a combination of RNA interference, complex affinity purification, and yeast two-hybrid approaches, we show here that the RNase PH homologues are important for maintenance of complex integrity. In contrast, the S1 domain proteins are not required for complex stability, although they are required for exosome function. Our results are partially consistent with the proposed model of the exosome, but indicate a different arrangement of the RNase PH proteins.     

Components of the SWI/SNF complex are required for asymmetric cell division in C. elegans

Asymmetric cell division is a fundamental process that produces cellular diversity during development. We have identified two mutants in C. elegans (psa-1 and psa-4) in which the asymmetry of T cell division is disrupted. psa-1 and psa-4 encode homologs of yeast SWI3 and SWI2/SNF2, respectively, which are components of the SWI/SNF complex. We show by RNA interference assay that homologs of other components of SWI/SNF are also involved in T cell division. psa-1 and psa-4 are likely to be required in the T cell during mitosis to cause asymmetric cell division. Because the SWI/SNF complex is required for asymmetric division in S. cerevisiae, these results demonstrate that at least some aspects of the mechanism of asymmetric cell division are conserved between yeast and a multicellular organism.     

Histone deacetylases in Trypanosoma brucei: two are essential and another is required for normal cell cycle progression

Reversible protein acetylation is established as a modification of major regulatory significance. In particular, histone acetylation regulates access to genetic information in eukaryotes. For example, class I and class II histone deacetylases are regulatory components of corepressor complexes involved in cell cycle progression and differentiation. Here, we have investigated the function of such enzymes in Trypanosoma brucei, mono-flagellated parasitic protozoa that branched very early from the eukaryotic lineage. Four T. brucei genes encoding histone deacetylase orthologues have been identified, cloned and characterized. The predicted deacetylases, DAC1-4 are approximately 43, 61, 75 and 64 kDa respectively. They share significant similarity with mammalian and yeast class I (DAC1 and DAC2) and class II (DAC3 and DAC4) histone deacetylases, and all except DAC2 have the critical residues predicted to be required for deacetylase activity. In gene targeting experiments, DAC1 and DAC3 appear to be essential whereas DAC2 and DAC4 are not required for viability. Of the two mutant cell types, the dac4 mutant displays a delay in the G2/M phase of the cell cycle. Our results provide genetic validation of DAC1 and DAC3 as potential chemotherapy targets and demonstrate that T. brucei expresses at least three probable histone deacetylases with distinct function.     

Characterization of a novel association between two trypanosome-specific proteins and 5S rRNA

P34 and P37 are two previously identified RNA binding proteins in the flagellate protozoan Trypanosoma brucei. RNA interference studies have determined that the proteins are essential and are involved in ribosome biogenesis. Here, we show that these proteins interact in vitro with the 5S rRNA with nearly identical binding characteristics in the absence of other cellular factors. The T. brucei 5S rRNA has a complex secondary structure and presents four accessible loops (A to D) for interactions with RNA-binding proteins. In other eukaryotes, loop C is bound by the L5 ribosomal protein and loop A mainly by TFIIIA. The binding of P34 and P37 to T. brucei 5S rRNA involves the LoopA region of the RNA, but these proteins also protect the L5 binding site located on LoopC.     

EXOG, a novel paralog of Endonuclease G in higher eukaryotes

Evolutionary conserved mitochondrial nucleases are involved in programmed cell death and normal cell proliferation in lower and higher eukaryotes. The endo/exonuclease Nuc1p, also termed 'yeast Endonuclease G (EndoG)', is a member of this class of enzymes that differs from mammalian homologs by the presence of a 5'-3' exonuclease activity in addition to its broad spectrum endonuclease activity. However, this exonuclease activity is thought to be essential for a function of the yeast enzyme in DNA recombination and repair. Here we show that higher eukaryotes in addition to EndoG contain its paralog 'EXOG', a novel EndoG-like mitochondrial endo/exonuclease. We find that during metazoan evolution duplication of an ancestral nuclease gene obviously generated the paralogous EndoG- and EXOG-protein subfamilies in higher eukaryotes, thereby maintaining the full endo/exonuclease activity found in mitochondria of lower eukaryotes. We demonstrate that human EXOG is a dimeric mitochondrial enzyme that displays 5'-3' exonuclease activity and further differs from EndoG in substrate specificity. We hypothesize that in higher eukaryotes the complementary enzymatic activities of EndoG and EXOG probably together account for both, the lethal and vital functions of conserved mitochondrial endo/exonucleases.     

Mitochondrial preprotein translocase of trypanosomatids has a bacterial origin

Mitochondria are found in all eukaryotic cells and derive from a bacterial endosymbiont [1, 2]. The evolution of a protein import system was a prerequisite for the conversion of the endosymbiont into a true organelle. Tom40, the essential component of the protein translocase of the outer membrane, is conserved in mitochondria of almost all eukaryotes but lacks bacterial orthologs [3-6]. It serves as the gateway through which all mitochondrial proteins are imported. The parasitic protozoa Trypanosoma brucei and its relatives do not have a Tom40-like protein, which raises the question of how proteins are imported by their mitochondria [7, 8]. Using a combination of bioinformatics and in vivo and in vitro studies, we have discovered that T. brucei likely employs a different import channel, termed ATOM (archaic translocase of the outer mitochondrial membrane). ATOM mediates the import of nuclear-encoded proteins into mitochondria and is essential for viability of trypanosomes. It is not related to Tom40 but is instead an ortholog of a subgroup of the Omp85 protein superfamily that is involved in membrane translocation and insertion of bacterial outer membrane proteins [9]. This suggests that the protein import channel in trypanosomes is a relic of an archaic protein transport system that was operational in the ancestor of all eukaryotes.     

Functions of the exosome in rRNA, snoRNA and snRNA synthesis.

The yeast nuclear exosome contains multiple 3'-->5' exoribonucleases, raising the question of why so many activities are present in the complex. All components are required during the 3' processing of the 5.8S rRNA, together with the putative RNA helicase Dob1p/Mtr4p. During this processing three distinct steps can be resolved, and hand-over between different exonucleases appears to occur at least twice. 3' processing of snoRNAs (small nucleolar RNAs) that are excised from polycistronic precursors or from mRNA introns is also a multi-step process that involves the exosome, with final trimming specifically dependent on the Rrp6p component. The spliceosomal U4 snRNA (small nuclear RNA) is synthesized from a 3' extended precursor that is cleaved by Rnt1p at sites 135 and 169 nt downstream of the mature 3' end. This cleavage is followed by 3'-->5' processing of the pre-snRNA involving the exosome complex and Dob1p. The exosome, together with Rnt1p, also participates in the 3' processing of the U1 and U5 snRNAs. We conclude that the exosome is involved in the processing of many RNA substrates and that different components can have distinct functions.     

An easily dissociated 26 S proteasome catalyzes an essential ubiquitin-mediated protein degradation pathway in Trypanosoma brucei

The 26 S proteasome, a complex between the 20 S proteasome and 19 S regulatory units, catalyzes ATP-dependent degradation of unfolded and ubiquitinated proteins in eukaryotes. We have identified previously 20 S and activated 20 S proteasomes in Trypanosoma brucei, but not 26 S proteasome. However, the presence of 26 S proteasome in T. brucei was suggested by the hydrolysis of casein by cell lysate, a process that requires ATP but is inhibited by lactacystin, and the lactacystin-sensitive turnover of ubiquitinated proteins in the intact cells. T. brucei cDNAs encoding the six proteasome ATPase homologues (Rpt) were cloned and expressed. Five of the six T. brucei Rpt cDNAs, except for Rpt2, were capable of functionally complementing the corresponding rpt deletion mutants of Saccharomyces cerevisiae. Immunoblots showed the presence in T. brucei lysate of the Rpt proteins, which co-fractionated with the yeast 19 S proteasome complex by gel filtration and localized in the 19 S fraction of a glycerol gradient. All the Rpt and putative 19 S non-ATPase (Rpn) proteins were co-immunoprecipitated from T. brucei lysate by individual anti-Rpt antibodies. Treatment of T. brucei cells with a chemical cross-linker resulted in co-immunoprecipitation of 20 S proteasome with all the Rpt and Rpn proteins that sedimented in a glycerol gradient to the position of 26 S proteasome. These data demonstrate the presence of 26 S proteasome in T. brucei cells, which apparently dissociate into 19 S and 20 S complexes upon cell lysis. RNA interference to block selectively the expression of proteasome 20 S core and Rpt subunits resulted in significant accumulation of ubiquitinated proteins accompanied by cessation of cell growth. Expression of yeast RPT2 gene in T. brucei Rpt2-deficient cells could not rescue the lethal phenotype, thus confirming the incompatibility between the two Rpt2s. The T. brucei 11 S regulator (PA26)-deficient RNA interference cells grew normally, suggesting the dispensability of activated 20 S proteasome in T. brucei.     

Stem-loop silencing reveals that a third mitochondrial DNA polymerase, POLID, is required for kinetoplast DNA replication in trypanosomes

Kinetoplast DNA (kDNA), the mitochondrial genome of trypanosomes, is a catenated network containing thousands of minicircles and tens of maxicircles. The topological complexity dictates some unusual features including a topoisomerase-mediated release-and-reattachment mechanism for minicircle replication and at least six mitochondrial DNA polymerases (Pols) for kDNA transactions. Previously, we identified four family A DNA Pols from Trypanosoma brucei with similarity to bacterial DNA Pol I and demonstrated that two (POLIB and POLIC) were essential for maintaining the kDNA network, while POLIA was not. Here, we used RNA interference to investigate the function of POLID in procyclic T. brucei. Stem-loop silencing of POLID resulted in growth arrest and the progressive loss of the kDNA network. Additional defects in kDNA replication included a rapid decline in minicircle and maxicircle abundance and a transient accumulation of minicircle replication intermediates before loss of the kDNA network. These results demonstrate that POLID is a third essential DNA Pol required for kDNA replication. While other eukaryotes utilize a single DNA Pol (Pol gamma) for replication of mitochondrial DNA, T. brucei requires at least three to maintain the complex kDNA network.     

The natural history of Get3‐like chaperones

Get3 in yeast or TRC40 in mammals is an ATPase that, in eukaryotes, is a central element of the GET or TRC pathway involved in the targeting of tail‐anchored proteins. Get3 has also been shown to possess chaperone holdase activity. A bioinformatic assessment was performed across all domains of life on functionally important regions of Get3 including the TRC40‐insert and the hydrophobic groove essential for tail‐anchored protein binding. We find that such a hydrophobic groove is much more common in bacterial Get3 homologs than previously appreciated based on a directed comparison of bacterial ArsA and yeast Get3. Furthermore, our analysis shows that the region containing the TRC40‐insert varies in length and methionine content to an unexpected extent within eukaryotes and also between different phylogenetic groups. In fact, since the TRC40‐insert is present in all domains of life, we suggest that its presence does not automatically predict a tail‐anchored protein targeting function. This opens up a new perspective on the function of organellar Get3 homologs in plants which feature the TRC40‐insert but have not been demonstrated to function in tail‐anchored protein targeting. Our analysis also highlights a large diversity of the ways Get3 homologs dimerize. Thus, based on the structural features of Get3 homologs, these proteins may have an unexplored functional diversity in all domains of life.      

A role for the exosome in the in vivo degradation of unstable mRNAs

In mammals, the mRNAs encoding many proteins involved in inflammation bear destabilizing AU-rich elements (AREs) in the 3'-untranslated region. The exosome, a complex of 3' --> 5' exonucleases, is rate limiting in the destruction of such mRNAs in a mammalian in vitro system, but a role in vivo has not been demonstrated. The phenomenon of ARE-mediated degradation also occurs in the protist parasite Trypanosoma brucei. Messenger RNAs with 3'-untranslated region U-rich elements, which strongly resemble AREs, are extremely unstable in the trypanosome form that parasitizes mammals. The first step in degradation of these mRNAs in vivo is rapid destruction of the 3'-untranslated region; subsequently the mRNA is destroyed by exonucleases acting in both 5' --> 3' and 3' --> 5' directions. We here investigated the roles of three subunits of the trypanosome exosome complex, RRP45, RRP4, and CSL4, in this process, depleting the individual subunits in vivo by inducible RNA interference. RRP45 depletion, which probably disrupts exosome integrity, caused a delay in the onset of degradation of the very unstable RNAs, but did not affect degradation of more stable species. Depletion of RRP4 or CSL4 does not affect the stability of the residual exosome and did not change mRNA degradation kinetics. We conclude that the exosome is required for the initiation of rapid degradation of unstable mRNAs in trypanosomes.     

Poly(A) tail-dependent exonuclease AtRrp41p from Arabidopsis thaliana rescues 5.8 S rRNA processing and mRNA decay defects of the yeast ski6 mutant and is found in an exosome-sized complex in plant and yeast cells

Eukaryotic 3'-->5' exonucleolytic activities are essential for a wide variety of reactions of RNA maturation and metabolism, including processing of rRNA, small nuclear RNA, and small nucleolar RNA, and mRNA decay. Two related but distinct forms of a complex containing 10 3'-->5' exonucleases, the exosome, are found in yeast nucleus and cytoplasm, respectively, and related complexes exist in human cells. Here we report on the characterization of the AtRrp41p, an Arabidopsis thaliana homolog of the Saccharomyces cerevisiae exosome subunit Rrp41p (Ski6p). Purified recombinant AtRrp41p displays a processive phosphorolytic exonuclease activity and requires a single-stranded poly(A) tail on a substrate RNA as a "loading pad." The expression of the Arabidopsis RRP41 cDNA in yeast rescues the 5.8 S rRNA processing and 3'-->5' mRNA degradation defects of the yeast ski6-100 mutant. However, neither of these defects can explain the conditional lethal phenotype of the ski6-100 strain. Importantly, AtRrp41p shares additional function(s) with the yeast Rrp41p which are essential for cell viability because it also rescues the rrp41 (ski6) null mutant. AtRrp41p is found predominantly in a high molecular mass complex in Arabidopsis and in yeast cells, and it interacts in vitro with the yeast Rrp44p and Rrp4p exosome subunits, suggesting that it can participate in evolutionarily conserved interactions that could be essential for the integrity of the exosome complex.     

Comparative Multiplexed Mass Spectrometric Analyses of Endogenously Expressed Yeast Nuclear and Cytoplasmic Exosomes

Here we combined tandem affinity purification with several mass-spectrometry-based approaches to gain more insight into the composition and structure of the yeast nuclear-cytoplasmic exosome protein complex. The yeast exosome fulfills several different functions in RNA metabolism and can be localized in both the cytoplasm and the nucleus. These two exosome complexes differ in protein composition, although they share several constituents. We focused on these differences in composition by selecting a nuclear-specific exosome protein (Rrp6) and a cytoplasmic-specific protein (Ski7) as the tandem-affinity-purification-tagged affinity bait protein. First, we investigated both these purified exosome assemblies by macromolecular mass spectrometry (MS) to determine the stability and mass of the intact protein complexes and to obtain information on composition and core constituents. We used tandem MS on these intact protein complexes to further probe the composition and to obtain insight into the peripheral nature of some of the constituents. Finally, we combine stable isotope labeling with MS to quantitate differences in exosome composition and posttranslational modifications. We identified a few phosphorylation sites that are differentially regulated between the cytoplasmic exosome and the nuclear exosome. From all of these data, we conclude that the yeast nuclear exosome and the cytoplasmic exosome share a common stable core complex, but are decorated with quite a few differing peripheral proteins. We show that the nuclear exosome selectively copurifies with the α/β importin heterodimer, which is known to be involved in the transport of proteins across the nuclear membrane.     

Arabidopsis homolog of the yeast TREX‐2 mRNA export complex: components and anchoring nucleoporin

Nuclear pore complexes (NPCs) are vital to nuclear–cytoplasmic communication in eukaryotes. The yeast NPC‐associated TREX‐2 complex, also known as the Thp1–Sac3–Cdc31–Sus1 complex, is anchored on the NPC via the nucleoporin Nup1, and is essential for mRNA export. Here we report the identification and characterization of the putative Arabidopsis thaliana TREX‐2 complex and its anchoring nucleoporin. Physical and functional evidence support the identification of the Arabidopsis orthologs of yeast Thp1 and Nup1. Of three Arabidopsis homologs of yeast Sac3, two are putative TREX‐2 components, but, surprisingly, none are required for mRNA export as they are in yeast. Physical association of the two Cdc31 homologs, but not the Sus1 homolog, with the TREX‐2 complex was observed. In addition to identification of these TREX‐2 components, direct interactions of the Arabidopsis homolog of DSS1, which is an established proteasome component in yeast and animals, with both the TREX‐2 complex and the proteasome were observed. This suggests the possibility of a link between the two complexes. Thus this work has identified the putative Arabidopsis TREX‐2 complex and provides a foundation for future studies of nuclear export in Arabidopsis.     

Yeast RNA exosome activity is necessary for maintaining cell wall stability through proper protein glycosylation

Nuclear RNA exosome is the main 3′→5′ RNA degradation and processing complex in eukaryotic cells and its dysregulation therefore impacts gene expression and viability. In this work we show that RNA exosome activity is necessary for maintaining cell wall stability in yeast Saccharomyces cerevisiae. While the essential RNA exosome catalytic subunit Dis3 provides exoribonuclease catalytic activity, the second catalytic subunit Rrp6 has a noncatalytic role in this process. RNA exosome cofactors Rrp47 and Air1/2 are also involved. RNA exosome mutants undergo osmoremedial cell lysis at high temperature or at physiological temperature upon treatment with cell wall stressors. Finally, we show that a defect in protein glycosylation is a major reason for cell wall instability of RNA exosome mutants. Genes encoding enzymes that act in the early steps of the protein glycosylation pathway are down-regulated at high temperature in cells lacking Rrp6 protein or Dis3 exoribonuclease activity and overexpression of the essential enzyme Psa1, that catalyzes synthesis of the mannosylation precursor, suppresses temperature sensitivity and aberrant morphology of these cells. Furthermore, this defect is connected to a temperature-dependent increase in accumulation of noncoding RNAs transcribed from loci of relevant glycosylation-related genes.