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Parmil K. Bansal Amanda Nourse Rashid Abdulle Katsumi Kitagawa 《The Journal of biological chemistry》2009,284(6):3586-3592
The kinetochore, which consists of DNA sequence elements and structural
proteins, is essential for high-fidelity chromosome transmission during cell
division. In budding yeast, Sgt1 and Hsp90 help assemble the core kinetochore
complex CBF3 by activating the CBF3 components Skp1 and Ctf13. In this study,
we show that Sgt1 forms homodimers by performing in vitro and in
vivo immunoprecipitation and analytical ultracentrifugation analyses.
Analyses of the dimerization of Sgt1 deletion proteins showed that the
Skp1-binding domain (amino acids 1–211) contains the Sgt1
homodimerization domain. Also, the Sgt1 mutant proteins that were unable to
dimerize also did not bind Skp1, suggesting that Sgt1 dimerization is
important for Sgt1-Skp1 binding. Restoring dimerization activity of a
dimerization-deficient sgt1 mutant (sgt1-L31P) by using the
CENP-B (centromere protein-B) dimerization
domain suppressed the temperature sensitivity, the benomyl sensitivity, and
the chromosome missegregation phenotype of sgt1-L31P. These results
strongly suggest that Sgt1 dimerization is required for kinetochore
assembly.Spindle microtubules are coupled to the centromeric region of the
chromosome by a structural protein complex called the kinetochore
(1,
2). The kinetochore is thought
to generate a signal that arrests cells during mitosis when it is not properly
attached to microtubules, thereby preventing aberrant chromosome transmission
to the daughter cells, which can lead to tumorigenesis
(3,
4). The kinetochore of the
budding yeast Saccharomyces cerevisiae has been characterized
thoroughly, genetically and biochemically; thus, its molecular structure is
the most well detailed to date. More than 70 different proteins comprise the
budding yeast kinetochore, and several of those are conserved in mammals
(2).The budding yeast centromere DNA is a 125-bp region that contains three
conserved regions, CDEI, CDEII, and CDEIII
(5,
6). CDEI is bound by Cbf1
(7–9).
CDEIII (25 bp) is essential for centromere function
(10) and is the site where
CBF3 binds to centromeric DNA. CBF3 contains four proteins: Ndc10, Cep3, Ctf13
(11–18),
and Skp1 (17,
18), all of which are
essential for viability. Mutations in any of the four CBF3 proteins abolish
the ability of CDEIII to bind to CBF3
(19,
20). All of the described
kinetochore proteins, except the CDEI-binding Cbf1, localize to kinetochores
dependent on the CBF3 complex
(2). Therefore, the CBF3
complex is the fundamental structure of the kinetochore, and the mechanism of
CBF3 assembly is of major interest.We previously isolated SGT1, the skp1-4
kinetochore-defective mutant dosage suppressor
(21). Sgt1 and Skp1 activate
Ctf13; thus, they are required for assembly of the CBF3 complex
(21). The molecular chaperone
Hsp90 is also required for the formation of the Skp1-Ctf13 complex
(22). Sgt1 has two highly
conserved motifs that are required for protein-protein interaction, the
tetratricopeptide repeat
(TPR)2
(21) and the CS
(CHORD protein- and Sgt1-specific) motif. We and others
(23–26)
have found that both domains are important for the interaction with Hsp90. The
Sgt1-Hsp90 interaction is required for the assembly of the core kinetochore
complex; this interaction is an initial step in kinetochore assembly
(24,
26,
27) that is conserved between
yeast and humans (28,
29).In this study, we further characterized the molecular mechanism of this
assembly process. We found that Sgt1 forms dimers in vivo, and our
results strongly suggest that Sgt1 dimerization is required for kinetochore
assembly in budding yeast. 相似文献
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Zdeněk Paris Piya Changmai Mary Anne T. Rubio Alena Zíková Kenneth D. Stuart Juan D. Alfonzo Julius Luke? 《The Journal of biological chemistry》2010,285(29):22394-22402
Fe/S clusters are part of the active site of many enzymes and are essential for cell viability. In eukaryotes the cysteine desulfurase Nfs (IscS) donates the sulfur during Fe/S cluster assembly and was thought sufficient for this reaction. Moreover, Nfs is indispensable for tRNA thiolation, a modification generally required for tRNA function and protein synthesis. Recently, Isd11 was discovered as an integral part of the Nfs activity at an early step of Fe/S cluster assembly. Here we show, using a combination of genetic, molecular, and biochemical approaches, that Isd11, in line with its strong association with Nfs, is localized in the mitochondrion of T. brucei. In addition to its involvement in Fe/S assembly, Isd11 also partakes in both cytoplasmic and mitochondrial tRNA thiolation, whereas Mtu1, another protein proposed to collaborate with Nfs in tRNA thiolation, is required for this process solely within the mitochondrion. Taken together these data place Isd11 at the center of these sulfur transactions and raises the possibility of a connection between Fe/S metabolism and protein synthesis, helping integrate two seemingly unrelated pathways. 相似文献
5.
Michelle Y. S. Shih Maureen A. Kane Ping Zhou C. L. Eric Yen Ryan S. Streeper Joseph L. Napoli Robert V. Farese Jr. 《The Journal of biological chemistry》2009,284(7):4292-4299
Retinoic acid (RA) is a potent signaling molecule that is essential for
many biological processes, and its levels are tightly regulated by mechanisms
that are only partially understood. The synthesis of RA from its precursor
retinol (vitamin A) is an important regulatory mechanism. Therefore, the
esterification of retinol with fatty acyl moieties to generate retinyl esters,
the main storage form of retinol, may also regulate RA levels. Here we show
that the neutral lipid synthesis enzyme acyl-CoA:diacylglycerol
acyltransferase 1 (DGAT1) functions as the major acyl-CoA:retinol
acyltransferase (ARAT) in murine skin. When dietary retinol is abundant, DGAT1
deficiency results in elevated levels of RA in skin and cyclical hair loss;
both are prevented by dietary retinol deprivation. Further, DGAT1-deficient
skin exhibits enhanced sensitivity to topically administered retinol. Deletion
of the enzyme specifically in the epidermis causes alopecia, indicating that
the regulation of RA homeostasis by DGAT1 is autonomous in the epidermis.
These findings show that DGAT1 functions as an ARAT in the skin, where it acts
to maintain retinoid homeostasis and prevent retinoid toxicity. Our findings
may have implications for human skin or hair disorders treated with agents
that modulate RA signaling.Regulation of cellular proliferation and differentiation of epithelial
tissues is crucial in embryonic development and in adult homeostasis. Retinoic
acid (RA)2 is a major
regulator of these processes
(1) through its ability to
serve as a ligand for RA nuclear receptors (RARs)
(2). Since RA is such a potent
signaling molecule, its levels must be tightly controlled. Indeed, excess RA
is highly teratogenic during embryonic development and may be toxic to adult
tissues (3). Further, RA is
used therapeutically for skin disorders, such as acne and psoriasis, and
certain cancers (4), but its
uses are often limited by local and systemic toxicity. Thus, understanding how
RA levels are regulated has important biological and clinical relevance.The synthesis of RA from its precursor retinol, or vitamin A, is a major
node in the regulation of RA levels
(5). To generate RA, retinol is
oxidized in two sequential reactions, catalyzed by retinol and retinal
dehydrogenases (5), whose
activities regulate RA homeostasis. We hypothesized that the availability of
retinol for these reactions may also be regulated by the balance between
retinol and retinyl esters. Indeed, the majority of retinol in the body is
stored as retinyl esters, which are concentrated in cytosolic lipid droplets
of cells and serve as a local source of retinol. Retinyl esters are also
stored in major organs, such as liver and white adipose tissue (WAT), from
which retinol can be mobilized to supply other tissues during increased
demand. Thus, retinol esterification may participate in regulating the retinol
pool available for RA synthesis.Retinol esterification is carried out by two distinct enzymatic activities.
One is mediated by lecithin:retinol acyltransferase (LRAT), which catalyzes
the covalent joining of a fatty acyl moiety from lecithin
(phosphatidylcholine) to retinol that is bound to cellular retinol-binding
protein (CRBP) (6,
7). LRAT activity is crucial
for maintaining tissue retinol stores. LRAT-null (Lrat-/-)
mice have severe reductions in hepatic and lung retinyl ester levels
(8–10),
which are accompanied by testicular hypoplasia/atrophy
(9) and blindness
(8). Retinyl ester levels are
normal in WAT and several other tissues, indicating alternative mechanisms for
retinol esterification (9,
10). This esterification is
probably mediated in part by acyl CoA:retinol acyltransferase (ARAT) enzymes,
which use fatty acyl-CoA and unbound retinol as substrates
(11). Although many tissues
exhibit ARAT activity (12),
attempts to purify and clone an ARAT gene were unsuccessful, and thus
molecular tools to study ARAT activity have been lacking. However, the enzyme
encoded by Dgat1, an acyl CoA:diacylglycerol acyltransferase (DGAT),
was recently reported to catalyze the ARAT reaction in vitro
(13,
14). Moreover, several tissues
of Dgat1-/- mice had reduced ARAT activity, and retinol
esterification was reduced in cultured murine embryonic fibroblasts lacking
DGAT1 (14). Most recently, a
study of Dgat1-/- mice demonstrated a role for the enzyme
in retinol absorption in the small intestine
(15). Thus, accumulating
evidence indicates that the retinol esterification activity of DGAT1 is of
biological, and possibly clinical, importance.In the current study, we investigated whether retinol esterification by
DGAT1 is important in murine skin. Dgat1-/- mice exhibit a
pleiotropic phenotype, which includes resistance to diet-induced obesity and
altered energy metabolism but also includes prominent phenotypic findings in
the skin
(16–19).
Retinoids play key roles in skin and hair biology
(20), and excess retinoids
induce epidermal hyperplasia, inhibit sebocyte proliferation and
differentiation, and alter hair growth
(21). Notably, the skin
manifestations of Dgat1-/- mice, which include alopecia
and sebaceous gland atrophy
(18), resemble those of
retinoid toxicity (22,
23). Thus, we hypothesized
that DGAT1 functions as an ARAT in murine skin and that the absence of DGAT1
alters retinoid homeostasis. In this study, we tested this hypothesis by
examining retinoid metabolism in the skin of DGAT1-deficient mice. 相似文献
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Nonstop mRNAs pose a challenge for bacteria, because translation cannot terminate efficiently without a stop codon. The trans-translation pathway resolves nonstop translation complexes by removing the nonstop mRNA, the incomplete protein, and the stalled ribosome. P1 co-transduction experiments demonstrated that tmRNA, a key component of the trans-translation pathway, is essential for viability in Shigella flexneri. tmRNA was previously shown to be dispensable in the closely related species Escherichia coli, because E. coli contains a backup system for trans-translation mediated by the alternative release factor ArfA. Genome sequence analysis showed that S. flexneri does not have a gene encoding ArfA. E. coli ArfA could suppress the requirement for tmRNA in S. flexneri, indicating that tmRNA is essential in S. flexneri because there is no functional backup system. These data suggest that resolution of nonstop translation complexes is required for most bacteria. 相似文献
10.
Elena B. M. Breidenstein Laure Janot Janine Strehmel Lucia Fernandez Patrick K. Taylor Irena Kukavica-Ibrulj Shaan L. Gellatly Roger C. Levesque Joerg Overhage Robert E. W. Hancock 《PloS one》2012,7(11)
Pseudomonas aeruginosa PAO1 lon mutants are supersusceptible to ciprofloxacin, and exhibit a defect in cell division and in virulence-related properties, such as swarming, twitching and biofilm formation, despite the fact that the Lon protease is not a traditional regulator. Here we set out to investigate the influence of a lon mutation in a series of infection models. It was demonstrated that the lon mutant had a defect in cytotoxicity towards epithelial cells, was less virulent in an amoeba model as well as a mouse acute lung infection model, and impacted on in vivo survival in a rat model of chronic infection. Using qRT-PCR it was demonstrated that the lon mutation led to a down-regulation of Type III secretion genes. The Lon protease also influenced motility and biofilm formation in a mucin-rich environment. Thus alterations in several virulence-related processes in vitro in a lon mutant were reflected by defective virulence in vivo. 相似文献
11.
Penicillin-Binding Protein 1 of Staphylococcus aureus Is Essential for Growth 总被引:3,自引:0,他引:3 下载免费PDF全文
pbpA, a gene encoding penicillin-binding protein (PBP) 1 of Staphylococcus aureus, was cloned in an Escherichia coli MC1061 transformant which grew on a plate containing 512 μg of vancomycin per ml. This gene encodes a 744-amino-acid sequence which conserves three motifs of PBPs, SXXK, SXN, and KTG. The chromosomal copy of pbpA could be disrupted only when RN4220, a methicillin-sensitive S. aureus strain, had additional copies of pbpA in its episome. Furthermore, these episomal copies of pbpA could not be eliminated by an incompatible plasmid when the chromosomal copy of pbpA was disrupted beforehand. Based on these observations, we concluded that pbpA is essential for the growth of methicillin-sensitive S. aureus. 相似文献
12.
Hassan Hashimi Lindsay McDonald Eva St?íbrná Julius Luke? 《The Journal of biological chemistry》2013,288(37):26914-26925
Letm1 is a conserved protein in eukaryotes bearing energized mitochondria. Hemizygous deletion of its gene has been implicated in symptoms of the human disease Wolf-Hirschhorn syndrome. Studies almost exclusively performed in opisthokonts have attributed several roles to Letm1, including maintaining mitochondrial morphology, mediating either calcium or potassium/proton antiport, and facilitating mitochondrial translation. We address the ancestral function of Letm1 in the highly diverged protist and significant pathogen, Trypanosoma brucei. We demonstrate that Letm1 is involved in maintaining mitochondrial volume via potassium/proton exchange across the inner membrane. This role is essential in the vector-dwelling procyclic and mammal-infecting bloodstream stages as well as in Trypanosoma brucei evansi, a form of the latter stage lacking an organellar genome. In the pathogenic bloodstream stage, the mitochondrion consumes ATP to maintain an energized state, whereas that of T. brucei evansi also lacks a conventional proton-driven membrane potential. Thus, Letm1 performs its function in different physiological states, suggesting that ion homeostasis is among the few characterized essential pathways of the mitochondrion at this T. brucei life stage. Interestingly, Letm1 depletion in the procyclic stage can be complemented by exogenous expression of its human counterpart, highlighting the conservation of protein function between highly divergent species. Furthermore, although mitochondrial translation is affected upon Letm1 ablation, it is an indirect consequence of K+ accumulation in the matrix. 相似文献
13.
Nadine Hagen Paul P. Van Veldhoven Richard L. Proia Hyejung Park Alfred H. Merrill Jr. Gerhild van Echten-Deckert 《The Journal of biological chemistry》2009,284(17):11346-11353
Cerebellar granule cells from sphingosine 1-phosphate (S1P) lyase-deficient
mice were used to study the toxicity of this potent sphingolipid metabolite in
terminally differentiated postmitotic neurons. Based on earlier findings with
the lyase-stable, semi-synthetic, cis-4-methylsphingosine phosphate,
we hypothesized that accumulation of S1P above a certain threshold induces
neuronal apoptosis. The present studies confirmed this conclusion and further
revealed that for S1P to induce apoptosis in lyase-deficient neurons it must
also be produced by sphingosine-kinase2 (SK2). These conclusions are based on
the finding that incubation of lyase-deficient neurons with either sphingosine
or S1P results in a similar elevation in cellular S1P; however, only S1P
addition to the culture medium induces apoptosis. This was not due to S1P
acting on the S1P receptor but to hydrolysis of S1P to sphingosine that was
phosphorylated by the cells, as described before for
cis-4-methylsphingosine. Although the cells produced S1P from both
exogenously added sphingosine as well as sphingosine derived from exogenous
S1P, the S1P from these two sources were not equivalent, because the former
was primarily produced by SK1, whereas the latter was mainly formed by SK2 (as
also was cis-4-methylsphingosine phosphate), based on studies in
neurons lacking SK1 or SK2 activity. Thus, these investigations show that, due
to the existence of at least two functionally distinct intracellular origins
for S1P, exogenous S1P can be neurotoxic. In this model, S1P accumulated due
to a defective lyase, however, this cause of toxicity might also be important
in other cases, as illustrated by the neurotoxicity of
cis-4-methylsphingosine phosphate.Sphingosine 1-phosphate
(S1P)2 is a potent
lipid mediator that has been shown to regulate a wide range of physiological
processes, including proliferation, differentiation, motility, cytoskeleton
rearrangements, and calcium homeostasis
(1,
2). There is convincing
experimental evidence that this bioactive sphingolipid can act both
extracellularly, as a ligand for a family of five specific G protein-coupled
receptors, and inside the cells, as a second messenger
(3,
4). In most cell types
described so far, S1P and its metabolic precursor ceramide exert antagonistic
effects on cell survival with S1P being generally regarded as a survival
signal, whereas ceramide and sphingosine are generally toxic
(5,
6). Interestingly, generation
of sphingosine and S1P is generally thought to be dependent on the
availability of ceramide (7),
however, relatively high amounts of S1P are also present in blood, lymph, and
cerebrospinal fluid (8,
9) and may serve as additional
sources for some cells.More than a decade ago, we introduced the synthetic sphingosine analog
cis-4-methylsphingosine as a tool for studies of sphingoid base
metabolism and function (10).
When added to the culture medium, this analog is taken up and mainly
phosphorylated to the respective cis-4-methylsphingosine phosphate,
which accumulates intracellularly, because it is poorly cleaved (if at all) by
S1P lyase (10). Intriguingly,
this compound promoted proliferation of quiescent Swiss 3T3 fibroblasts
(11), as does S1P
(12), but induced apoptosis in
postmitotic terminally differentiated primary cultured neurons
(13).Despite the fact that neither S1P nor sphingosine were able to induce
apoptosis in neurons, we proposed that cis-4-methylsphingosine is
phosphorylated by cells yielding a metabolically stable analog of S1P. This
prediction was based on experimental results indicating that the different
physiological effects, apoptosis in the case of the accumulating metabolically
stable synthetic compound versus no apoptosis in the case of the
short living S1P, rely only on nuances of impact
(13). Both sphingoid
phosphates affected similar pathways. However, the effect of the synthetic
accumulated compound was more pronounced and persistent when compared with the
more transient and less pronounced effect of the short living physiological
counterpart (13). We therefore
assumed that conditions that allow sufficient accumulation of S1P in primary
cultured neurons should end up in neuronal apoptosis.To explore this hypothesis, which might be relevant to neurodegenerative
processes, we attempted to elevate intracellular S1P using siRNAs directed to
S1P lyase (encoded by the Sgpl1 gene). However, suppression of lyase
by ∼70% did not result in an accumulation of endogenous S1P in primary
cultured neurons (14).The central aim of the present study was to evaluate the hypothesis that
endogenous S1P induces neuronal apoptosis when it exceeds a certain threshold
by a more effective method for lyase activity suppression. We thus used
primary cultured neurons prepared from cerebella of 6-day-old lyase-deficient
mice (15). The present studies
not only confirmed that elevation of S1P induced cell death but also revealed
that the origin of the S1P was important. Intriguingly, neuronal apoptosis was
induced only by S1P derived from exogenous S1P that was dephosphorylated and
then resynthesized to S1P by sphingosine kinase 2 (SK2). Interestingly, we
then found that this is also the kinase responsible for synthesis of
cis-4-methylsphingosine phosphate. In addition, our data document
that the pro-apoptotic effect of S1P is independent of cellular ceramide
content. 相似文献
14.
The AT-Hook Protein D1 Is Essential for Drosophila melanogaster Development and Is Implicated in Position-Effect Variegation 总被引:2,自引:0,他引:2 下载免费PDF全文
Nathalie Aulner Caroline Monod Guillaume Mandicourt Denis Jullien Olivier Cuvier Alhousseynou Sall Sam Janssen Ulrich K. Laemmli Emmanuel Ks 《Molecular and cellular biology》2002,22(4):1218-1232
15.
Highlights? Ensconsin is required for cargo transport driven by kinesin-1 ? The C terminus of ensconsin activates kinesin-1; its N terminus binds microtubules ? Kinesin-1 mutants without autoinhibitory activity do not require ensconsin 相似文献
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Andreas Gewies Mercedes Castineiras-Vilarino Uta Ferch Nina J?hrling Katja Heinrich Ulrike Hoeckendorf Gerhard K. H. Przemeck Matthias Munding Olaf Gro? Timm Schroeder Marion Horsch E. Loraine Karran Aneela Majid Stefan Antonowicz Johannes Beckers Martin Hrabé de Angelis Hans-Ulrich Dodt Christian Peschel Irmgard F?rster Martin J. S. Dyer Jürgen Ruland 《PloS one》2013,8(11)
Members of the PRDM protein family have been shown to play important roles during embryonic development. Previous in vitro and in situ analyses indicated a function of Prdm6 in cells of the vascular system. To reveal physiological functions of Prdm6, we generated conditional Prdm6-deficient mice. Complete deletion of Prdm6 results in embryonic lethality due to cardiovascular defects associated with aberrations in vascular patterning. However, smooth muscle cells could be regularly differentiated from Prdm6-deficient embryonic stem cells and vascular smooth muscle cells were present and proliferated normally in Prdm6-deficient embryos. Conditional deletion of Prdm6 in the smooth muscle cell lineage using a SM22-Cre driver line resulted in perinatal lethality due to hemorrhage in the lungs. We thus identified Prdm6 as a factor that is essential for the physiological control of cardiovascular development. 相似文献
18.
人源NAP1L5为核小体组装蛋白(NAP-1)家族成员,目前功能未知。肝癌研究暗示Nap1l5可能为抑癌基因,抑制细胞增殖;但NAP-1家族其他功能已知的一些成员,可促进细胞增殖和加快周期进程。人源NAP1L5是促进还是抑制细胞增殖,目前未知。本研究里,我们发现过表达Nap1l5促进293T细胞增殖,抑制表达则降低293T细胞增殖速度。细胞周期分析表明:Nap1l5过表达增加G2期、减少G1期细胞比例;抑制Nap1l5表达,则增加G1期、减少G2期细胞比例。我们的研究表明:人源NAP1L5会加快293T细胞周期进程,促进增殖,并且提示原来关于Nap1l5是抑癌基因的推测是不对的。 相似文献
19.
Nicolas Jaé Pingping Wang Tianpeng Gu Martin Hühn Zsofia Palfi Henning Urlaub Albrecht Bindereif 《Eukaryotic cell》2010,9(3):379-386
Spliceosomal small nuclear ribonucleoproteins (snRNPs) in trypanosomes contain either the canonical heptameric Sm ring or variant Sm cores with snRNA-specific Sm subunits. Here we show biochemically by a combination of RNase H cleavage and tandem affinity purification that the U4 snRNP contains a variant Sm heteroheptamer core in which only SmD3 is replaced by SSm4. This U4-specific, nuclear-localized Sm core protein is essential for growth and splicing. As shown by RNA interference (RNAi) knockdown, SSm4 is specifically required for the integrity of the U4 snRNA and the U4/U6 di-snRNP in trypanosomes. In addition, we demonstrate by in vitro reconstitution of Sm cores that under stringent conditions, the SSm4 protein suffices to specify the assembly of U4 Sm cores. Together, these data indicate that the assembly of the U4-specific Sm core provides an essential step in U4/U6 di-snRNP biogenesis and splicing in trypanosomes.The excision of intronic sequences from precursor mRNAs is a critical step during eukaryotic gene expression. This reaction is catalyzed by the spliceosome, a macromolecular complex composed of small nuclear ribonucleoproteins (snRNPs) and many additional proteins. Spliceosome assembly and splicing catalysis occur in an ordered multistep process, which includes multiple conformational rearrangements (35). Spliceosomal snRNPs are assembled from snRNAs and protein components, the latter of which fall into two classes: snRNP-specific and common proteins. The common or canonical core proteins are also termed Sm proteins, specifically SmB, SmD1, SmD2, SmD3, SmE, SmF, and SmG (10; reviewed in reference 9), which all share an evolutionarily conserved bipartite sequence motif (Sm1 and Sm2) required for Sm protein interactions and the formation of the heteroheptameric Sm core complex around the Sm sites of the snRNAs (3, 7, 29). Prior to this, the Sm proteins form three heteromeric subcomplexes: SmD3/SmB, SmD1/SmD2, and SmE/SmF/SmG (23; reviewed in reference 34). Individual Sm proteins or Sm subcomplexes cannot stably interact with the snRNA. Instead, a stable subcore forms by an association of the subcomplexes SmD1/SmD2 and SmE/SmF/SmG with the Sm site on the snRNA; the subsequent integration of the SmD3/SmB heterodimer completes Sm core assembly.In addition to the canonical Sm proteins, other proteins carrying the Sm motif have been identified for many eukaryotes. Those proteins, termed LSm (like Sm) proteins, exist in distinct heptameric complexes that differ in function and localization. For example, a complex composed of LSm1 to LSm7 (LSm1-7) accumulates in cytoplasmic foci and participates in mRNA turnover (4, 8, 31). Another complex, LSm2-8, binds to the 3′ oligo(U) tract of the U6 snRNA in the nucleus (1, 15, 24). Finally, in the U7 snRNP, which is involved in histone mRNA 3′-end processing, the Sm proteins SmD1 and SmD2 are replaced by U7-specific LSm10 and LSm11 proteins, respectively (20, 21; reviewed in reference 28).This knowledge is based primarily on the mammalian system, where spliceosomal snRNPs are biochemically well characterized (34). In contrast, for trypanosomes, comparatively little is known about the components of the splicing machinery and their assembly and biogenesis. In trypanosomes, the expression of all protein-encoding genes, which are arranged in long polycistronic units, requires trans splicing. Only a small number of genes are additionally processed by cis splicing (reviewed in reference 11). During trans splicing, a short noncoding miniexon, derived from the spliced leader (SL) RNA, is added to each protein-encoding exon. Regarding the trypanosomal splicing machinery, the U2, U4/U6, and U5 snRNPs are considered to be general splicing factors, whereas the U1 and SL snRNPs represent cis- and trans-splicing-specific components, respectively. In addition to the snRNAs, many protein splicing factors in trypanosomes have been identified based on sequence homology (for example, see references 14 and 19).Recent studies revealed variations in the Sm core compositions of spliceosomal snRNPs from Trypanosoma brucei. Specifically, in the U2 snRNP, two of the canonical Sm proteins, SmD3 and SmB, are replaced by two novel, U2 snRNP-specific proteins, Sm16.5K and Sm15K (33). In this case, an unusual purine nucleotide, interrupting the central uridine stretch of the U2 snRNA Sm site, discriminates between the U2-specific and the canonical Sm cores. A second case of Sm core variation was reported for the U4 snRNP, in which a single protein, SmD3, was suggested to be replaced by the U4-specific LSm protein initially called LSm2, and later called SSm4, based on a U4-specific destabilization after SSm4 knockdown (30). A U4-specific Sm core variation was also previously suggested and discussed by Wang et al. (33), based on the inefficient pulldown of U4 snRNA through tagged SmD3 protein. However, neither of these two studies conclusively demonstrated by biochemical criteria that the specific Sm protein resides in the U4 Sm core; a copurification of other snRNPs could not be unequivocally ruled out.By using a combination of RNase H cleavage, tandem affinity purification, and mass spectrometry, we provide here direct biochemical evidence that in the variant Sm core of the U4 snRNP, only SmD3 is replaced by the U4-specific SSm4. SSm4 is nuclear localized, and the silencing of SSm4 leads to a characteristic phenotype: dramatic growth inhibition, general trans- and cis-splicing defects, a loss of the integrity of the U4 snRNA, as well as a destabilization of the U4/U6 di-snRNP. Furthermore, in vitro reconstitution assays revealed that under stringent conditions, SSm4 is sufficient to specify U4-specific Sm core assembly. In sum, our data establish SSm4 as a specific component of the U4 Sm core and demonstrate its importance in U4/U6 di-snRNP biogenesis, splicing function, and cell viability. 相似文献
20.
Abdellah Barakate James D. Higgins Sebastian Vivera Jennifer Stephens Ruth M. Perry Luke Ramsay Isabelle Colas Helena Oakey Robbie Waugh F. Chris H. Franklin Susan J. Armstrong Claire Halpin 《The Plant cell》2014,26(2):729-740
In many cereal crops, meiotic crossovers predominantly occur toward the ends of
chromosomes and 30 to 50% of genes rarely recombine. This limits the exploitation of
genetic variation by plant breeding. Previous reports demonstrate that chiasma
frequency can be manipulated in plants by depletion of the synaptonemal complex
protein ZIPPER1 (ZYP1) but conflict as to the direction of change, with fewer
chiasmata reported in Arabidopsis thaliana and more crossovers
reported for rice (Oryza sativa). Here, we use RNA interference
(RNAi) to reduce the amount of ZYP1 in
barley (Hordeum vulgare) to only 2 to 17% of normal zygotene levels.
In the ZYP1RNAi lines, fewer than half of the chromosome
pairs formed bivalents at metaphase and many univalents were observed, leading to
chromosome nondisjunction and semisterility. The number of chiasmata per cell was
reduced from 14 in control plants to three to four in the ZYP1-depleted lines,
although the localization of residual chiasmata was not affected. DNA double-strand
break formation appeared normal, but the recombination pathway was defective at later
stages. A meiotic time course revealed a 12-h delay in prophase I progression to the
first labeled tetrads. Barley ZYP1 appears to function similarly to ZIP1/ZYP1 in
yeast and Arabidopsis, with an opposite effect on crossover number
to ZEP1 in rice, another member of the Poaceae. 相似文献