Context dependent splicing functions of Bud31/Ycr063w define its role in budding and cell cycle progression

The yeast Bud31 protein, a Prp19 complex (NTC) member, aids spliceosome assembly and thus promotes efficient pre-mRNA splicing. The bud31 null cells show mild budding abnormalities at optimal growth temperatures and, at higher temperatures, have growth defects with aberrant budding. Here we have assessed cell cycle transitions which require Bud31. We find Bud31 facilitates passage through G1-S regulatory point (Start) but is not needed for G2-M transition or for exit from mitosis. To co-relate Bud31 functions in cell division with splicing, we studied the splicing status of transcripts that encode proteins involved in budding. We find Bud31 promotes efficient splicing of only some of these pre-mRNAs, for example, ARP2 and SRC1. Wild type cells have a long and a short isoform of SRC1 mRNA and protein, out of which the shorter mRNA splice variant is predominant. bud31 Delta cells show inefficient SRC1 splicing and entirely lack the shorter SRC1 spliced mRNA isoform. Yeast PRP17, another NTC sub-complex member, is also required for G1-S and G2-M cell cycle transitions. We examined genetic interactions between BUD31 and PRP17. While both factors were needed for efficient cell cycle dependent gene expression, our data indicate that distinct pre-mRNAs depend on each of these non-essential splicing factors

Antimutagenic activity of compounds isolated from Ajuga bracteosa Wall ex. Benth against EMS induced mutagenicity in mice

Ajuga bracteosa Wall ex. Benth. (Lamiaceae) has been reported to possess many biological activities including antibacterial, antifungal, antispasmodic and antioxidant activity but there is no report as such on its mutagenic and/or anti-mutagenic activity. The aim of the present study was to isolate compounds from the methanol extract of the aerial parts of Ajuga bracteosa and determine their anti-mutagenic activity against the mutagen, EMS in animal model mice. The study was undertaken in order to corroborate the traditional use of the plant Ajuga bracteosa. The compounds were isolated from the methanol extract of the aerial parts of Ajuga bracteosa using silica gel column chromatography. Structural elucidation of the isolated compounds was done using spectral data analysis and comparison with literature. High performance liquid chromatography (HPLC) was used for the qualitative and quantitative determination of the isolated compounds in the crude methanol extract. The isolated compounds and standard drug were evaluated in vivo for antimutagenic activity against EMS induced mutagenicity taking mice as model organism by micronucleus and chromosomal aberration tests. Four major compounds were identified as 1) 14, 15-dihydroajugapitin 2) β- Sitosterol 3) Stigmasterol and 4) 8-O-acetylharpagide. A quick and sensitive HPLC method was developed for qualitative and quantitative determination of three isolated marker compounds from Ajuga bracteosa. 14, 15-dihydroajugapitin reduced the micronuclei by 85.10%, followed by β- Sitosterol (72.3%) while as 8-O-acetylharpagide reduced the micronuclei by 46%. It is therefore evident from the present study that the plant contains rich source of anticancer and antimutagenic drugs. Keywords: Ajuga bracteosa, HPLC, EMS, Mice, Antimutagenic activity, β-Sitostero

sMEK1 promotes crosstalk between IRE1 and Akt signaling pathways: Evidence for a novel IRE1/sMEK1/Akt complex

AbstractER is facilitated with a dynamic cellular pathway namely Unfolded Protein Response (UPR): an adaptive signalling mechanism that maintains proteostasis in response to ER stress. IRE1 is one of the three transmembrane sensors of UPR with dual protein kinase and ribonuclease activities. IRE1 acts as a central molecule of UPR, which associates with a number of proteins that either regulate its activity or connect it to other pathways. Here, we report sMEK1 and Akt as novel interacting partners of IRE1 which associate to orchestrate the IRE1 and Akt signalling networks. Our study revealed that ER stress negatively regulates Akt through IRE1 protein. We found that IRE1/sMEK1/Akt form a ternary complex, which results in the dephosphorylation of Akt by protein phosphotase sMEK1 in presence of activated IRE1. Together, this study highlights the UPR/Akt link by delineating the molecular mechanism along with giving insights into the overall impact of this interaction.</jats:p

Bid as a novel interacting partner of IRE1 differentially regulating its RNAse activity

AbstractUnfolded protein response is a dynamic signalling pathway, which is involved in the maintenance of proteostasis and cellular homeostasis. IRE1, a transmembrane signalling protein represents the start point of a highly conserved UPR signalling cascade. IRE1 is endowed with kinase and endoribonuclease activities. The activation of the kinase domain of IRE1 by trans-autophosphorylation leads to the activation of its RNAse domain. RNAse domain performs atypical splicing of Xbp1 mRNA and degradation of mRNAs by an effector function known as Regulated IRE1 Dependent Decay (RIDD). The regulation of the distinctive nature of the IRE1 ribonuclease function is potentially mediated by a dynamic protein structure UPRosome that is an assembly of a huge number of proteins on IRE1. Here, we reported that Bid is a novel recruit to UPRosome, which directly interacts with the cytoplasmic domain of IRE1. Bid controls the auto-phosphorylation of IRE1 in a negative manner where Bid overexpression conditions displayed reduced phosphorylation levels of IRE1 and Bid knockdown cells showed slightly enhanced IRE1 phosphorylation. This effect was reciprocated with JNK, a downstream target of IRE1. Our Insilico analysis revealed that Bid binding to IRE1 dimer averts its structural flexibility and thereby preventing its trans-autophosphorylation activity. We found that the effect of Bid is specific to the IRE1 branch of UPR signalling and competitive in nature. The highlighting observation of the study was that Bid stimulated a differential activity of the IRE1 RNAse domain towards Xbp1 splicing and RIDD. These results together establish that Bid is a part of the UPRosome and modulates IRE1 in a way to differentially regulate its RNAse outputs.</jats:p

Serpina1 hepatocyte-specific promoter polymorphism associate with chronic obstructive pulmonary disease in a study of kashmiri ancestry individuals

Different mutations in coding and non-coding sequences of the SERPINA1 gene have been implicated in the pathogenesis of COPD. However, - 10T/C mutation in the hepatocyte-directed promoter region has not been associated with COPD pathogenesis so far. Here, we report an increased frequency of - 10C genotype that is associated with decreased levels of serum alpha1-antitrypsin (alpha 1AT) in COPD patients. The quantification of serum alpha 1AT was done by ELISA, the phenol-chloroform method was used for DNA extraction, PCR products were directly sequenced. The IBM SPSS Statistics v21 software was used for statistical analyses of the data. The mean serum alpha 1AT level was found to be 1.203+0.239 and 3.162+0.160 g/L in COPD cases and in control, respectively. The - 10C allele is associated with an increased risk of COPD [OR, 3.50 (95%CI, 1.86-6.58); p < 0.001]. The combined variant genotype (TT+CC) was significantly found associated with an increased risk of COPD [OR, 3.20 (95% CI, 1.47-6.96); p = 0.003]. A significant association of the family history with COPD (overall p value= 0.0331) suggests that genetics may play an important role in the pathogenesis of COPD. The polymorphism associated with hepatocyte-specific promoter region (- 10T/C) is likely to be associated with the pathogenesis of COPD. It is quite possible that the change of the base in the hepatocyte-specific promoter of the SERPINA1 gene can modulate its strength, thereby driving the reduced expression of alpha 1AT.Department of Biotechnology (DBT), New Delhi: BT/PR7240/MED/30/915/2012; Department of Science and Technology: SB/SO/AS-126/2012; FIST: SR/FST/LSI-384/2008; SAP: F.3-26/2011(SAP-II); UGC: F.3-26/2011(SAP-II

Splicing Functions and Global Dependency on Fission Yeast Slu7 Reveal Diversity in Spliceosome Assembly

The multiple short introns in Schizosaccharomyces pombe genes with degenerate cis sequences and atypically positioned polypyrimidine tracts make an interesting model to investigate canonical and alternative roles for conserved splicing factors. Here we report functions and interactions of the S. pombe slu7(+) (spslu7(+)) gene product, known from Saccharomyces cerevisiae and human in vitro reactions to assemble into spliceosomes after the first catalytic reaction and to dictate 3' splice site choice during the second reaction. By using a missense mutant of this essential S. pombe factor, we detected a range of global splicing derangements that were validated in assays for the splicing status of diverse candidate introns. We ascribe widespread, intron-specific SpSlu7 functions and have deduced several features, including the branch nucleotide-to-3' splice site distance, intron length, and the impact of its A/U content at the 5' end on the intron's dependence on SpSlu7. The data imply dynamic substrate-splicing factor relationships in multiintron transcripts. Interestingly, the unexpected early splicing arrest in spslu7-2 revealed a role before catalysis. We detected a salt-stable association with U5 snRNP and observed genetic interactions with spprp1(+), a homolog of human U5-102k factor. These observations together point to an altered recruitment and dependence on SpSlu7, suggesting its role in facilitating transitions that promote catalysis, and highlight the diversity in spliceosome assembly