ISOLATION AND CHARACTERIZATION OF THE FOUR ARABIDOPSIS THALIANA POLY(A) POLYMERASE GENES

Poly(A) tail addition to pre-mRNAs is a highly coordinated and essential step in mRNA maturation involving multiple cis- and trans-acting factors. The trans-acting factor, poly(A) polymerase (PAP) plays an essential role in the polyadenylation of mRNA precursors. The Arabidopsis thaliana genome contains four putative PAP genes. We have found, using in silico analysis and transgenic plants expressing GUS under the control of the four PAP promoters, that each of these genes is expressed in overlapping, yet unique patterns. This gives rise to the possibility that these genes are not redundant and may be essential for plant survival. To further test this, inducible RNAi and T-DNA mutagenized plants were obtained and analyzed. Plants lacking all, or most, of each PAP gene product, due to RNAi induction, were not viable at any of the stages of plant growth tested. Furthermore, T-DNA PCR analysis determined that no plants containing a homozygous mutation, were viable. This data reveals that lack of any of the four PAP gene products has a significant effect on the plants ability of survive, thus indicating that each PAP gene is essential. Finally, transient expression experiments with each of the full length PAP cDNAs fused to GFP showed that the PAP I, PAP II and PAP IV gene products are localized throughout the nucleus and within nuclear speckles. The cellular localization of PAP III could not be determined

Epigenetic gene silencing by heterochromatin primes fungal resistance

Genes embedded in H3 lysine 9 methylation (H3K9me)–dependent heterochromatin are transcriptionally silenced. In fission yeast, Schizosaccharomyces pombe, H3K9me-mediated heterochromatin can be transmitted through cell division provided the counteracting demethylase Epe1 is absent. Under certain conditions wild-type cells might utilize heterochromatin heritability to form epimutations, phenotypes mediated by unstable silencing rather than DNA changes. This study shows that resistant heterochromatin-dependent epimutants arise in threshold levels of caffeine. Unstable resistant isolates exhibit distinct heterochromatin islands, which reduce expression of underlying genes, some of which confer resistance when mutated. Targeting synthetic heterochromatin to implicated loci confirms that resistance results from heterochromatin-mediated silencing. The analyses presented here reveal that epigenetic processes promote phenotypic plasticity, allowing wild-type cells to adapt to non-favorable environments without altering their genotype. In some isolates, subsequent or co-occurring gene amplification events augment resistance. Caffeine impacts two anti silencing factors: Epe1 levels are downregulated, reducing its chromatin association; and Mst2 histone acetyltransferase expression switches to a shortened isoform. Thus, heterochromatin-dependent epimutant formation provides a bet-hedging strategy that allows cells to remain genetically wild-type but adapt transiently to external insults. Unstable caffeine-resistant isolates show cross-resistance to antifungal agents, suggesting that related heterochromatin-dependent processes may contribute to antifungal drug resistance in plant and human pathogenic fungi

Deciphering the role of Hsp31 as a multitasking chaperone

Among different type of protein aggregation, amyloids are biochemically well characterized state of protein aggregation that is commonly associated with a large number of neurodegenerative diseases in mammals and cause heritable traits in Saccharomyces cerevisiae. Among many other neurodegenerative diseases linked with amyloids, Parkinson’s disease is the second most common disorder that is caused by progressive deterioration of dopaminergic neurons in substantia nigra. Cellular stresses such as accumulation of high level of reactive oxygen species, mitochondrial dysfunction and α-syn aggregation lead to toxicity and neuronal cell death in Parkinson’s disease patients. Mutations in certain genes are also involved in the development of a familial form of PD including PARK7 that encodes DJ-1. DJ-1 is a member of ThiJ/DJ-1/PfpI protein superfamily that are the quintessential multitasking or moonlighting protein family as evidenced by their involvement in multiple cellular functions including oxidative stress sensing, protein folding, proteasome degradation, mitochondrial complex stabilization, methylglyoxalase and deglycation enzyme activities. The members of the ThiJ/DJ-1/Pfp1 superfamily appear to have evolved to numerous mechanisms to manage cellular stress. The protein superfamily members are present across the evolutionary spectrum including prokaryotes and the budding yeast, S. cerevisiae, that has four paralogs Hsp31, Hsp32, Hsp33, and Hsp34. Hsp31 consists of 237 amino acids with a MW of 25.5 kDa and forms a homodimer in solution. It possesses the Cys-His-Glu catalytic triad common to ThiJ/DJ-1/PfpI superfamily proteins. Previously, we have shown that Hsp31 possesses chaperone properties with protective effects against α-syn toxicity in yeast. Recently, it is shown that Hsp31 has a methylglyoxalase activity that converts the toxic metabolite methylglyoxal into lactate. Here, we confirmed that Hsp31 is a robust methylglyoxalse that is more potent in activity than its human homolog DJ-1. We demonstrated that Hsp31 chaperone activity to protect the cells from α-syn toxicity is not under the influence of its enzymatic activity or autophagy pathway. Moreover, we confirmed that Hsp31 expression is induced by H2O2 mediated oxidative stress and further showed an increased expression of Hsp31 under α-syn mediated proteotoxic stress. These results establish that Hsp31 molecular chaperone activity is self-sufficient to protect the cells from stress conditions without requiring its enzymatic activities

The Schizosaccharomyces Pombe Pla1 Gene Encodes a Poly(A) Polymerase and Can Functionally Replace Its Saccharomyces Cerevisiae Homologue

We have isolated the poly(A) polymerase (PAP) encoding gene pla1 [for poly(A) polymerase] from the fission yeast Schizosaccharomyces pombe. Protein sequence alignments with other poly(A) polymerases reveal that pla1 is more closely related to Saccharomyces cerevisiae PAP than to bovine PAP. The two yeast poly(A) polymerases share significant sequence homology not only in the generally conserved N-terminal part but also in the C-terminus. Furthermore, pla1 rescues a S.cerevisiae PAP1 disruption mutant. An extract from the complemented strain is active in the specific in vitro polyadenylation assay. In contrast, recombinant PLA1 protein can not replace bovine PAP in the mammalian in vitro polyadenylation assay. These results indicate a high degree of conservation of the polyadenylation machinery among the evolutionary diverged budding and fission yeast

The Schizosaccharomyces pombe pla1 gene encodes a poly(A) polymerase and can functionally replace its Saccharomyces cerevisiae homologue.

We have isolated the poly(A) polymerase (PAP) encoding gene pla1 [for poly(A) polymerase] from the fission yeast Schizosaccharomyces pombe. Protein sequence alignments with other poly(A) polymerases reveal that pla1 is more closely related to Saccharomyces cerevisiae PAP than to bovine PAP. The two yeast poly(A) polymerases share significant sequence homology not only in the generally conserved N-terminal part but also in the C-terminus. Furthermore, pla1 rescues a S. cerevisiae PAP1 disruption mutant. An extract from the complemented strain is active in the specific in vitro polyadenylation assay. In contrast, recombinant PLA1 protein can not replace bovine PAP in the mammalian in vitro polyadenylation assay. These results indicate a high degree of conservation of the polyadenylation machinery among the evolutionary diverged budding and fission yeasts