Profiling of mismatch discrimination in RNAi enabled rational design of allele-specific siRNAs

Silencing specificity is a critical issue in the therapeutic applications of siRNA, particularly in the treatment of single nucleotide polymorphism (SNP) diseases where discrimination against single nucleotide variation is demanded. However, no generally applicable guidelines are available for the design of such allele-specific siRNAs. In this paper, the issue was approached by using a reporter-based assay. With a panel of 20 siRNAs and 240 variously mismatched target reporters, we first demonstrated that the mismatches were discriminated in a position-dependent order, which was however independent of their sequence contexts using position 4th, 12th and 17th as examples. A general model was further built for mismatch discrimination at all positions using 230 additional reporter constructs specifically designed to contain mismatches distributed evenly along the target regions of different siRNAs. This model was successfully employed to design allele-specific siRNAs targeting disease-causing mutations of PIK3CA gene at two SNP sites. Furthermore, conformational distortion of siRNA-target duplex was observed to correlate with the compromise of gene silencing. In summary, these findings could dramatically simplify the design of allele-specific siRNAs and might also provide guide to increase the specificity of therapeutic siRNAs.     

Contributions of the two accessory subunits,RNASEH2B and RNASEH2C,to the activity and properties of the human RNase H2 complex

Eukaryotic RNase H2 is a heterotrimeric enzyme. Here, we show that the biochemical composition and stoichiometry of the human RNase H2 complex is consistent with the properties previously deduced from genetic studies. The catalytic subunit of eukaryotic RNase H2, RNASEH2A, is well conserved and similar to the monomeric prokaryotic RNase HII. In contrast, the RNASEH2B and RNASEH2C subunits from human and Saccharomyces cerevisiae share very little homology, although they both form soluble B/C complexes that may serve as a nucleation site for the addition of RNASEH2A to form an active RNase H2, or for interactions with other proteins to support different functions. The RNASEH2B subunit has a PIP-box and confers PCNA binding to human RNase H2. Unlike Escherichia coli RNase HII, eukaryotic RNase H2 acts processively and hydrolyzes a variety of RNA/DNA hybrids with similar efficiencies, suggesting multiple cellular substrates. Moreover, of five analyzed mutations in human RNASEH2B and RNASEH2C linked to Aicardi-Goutières Syndrome (AGS), only one, R69W in the RNASEH2C protein, exhibits a significant reduction in specific activity, revealing a role for the C subunit in enzymatic activity. Near-normal activity of four AGS-related mutant enzymes was unexpected in light of their predicted impairment causing the AGS phenotype.     

Bombesin enhances TGF-β growth inhibitory effect through apoptosis induction in intestinal epithelial cells

Mammalian intestinal epithelium undergoes continuous cell turn over, with cell proliferation in the crypts and apoptosis in the villus. Both transforming growth factor (TGF)-β and gastrin-releasing peptide (GRP) are involved in the regulation of intestinal epithelial cells for division, differentiation, adhesion, migration and death. Previously, we have shown that TGF-β and bombesin (BBS) synergistically induce cyclooxygenase-2 (COX-2) expression and subsequent prostaglandin E₂ (PGE2) production through p38^MAPK in rat intestinal epithelial cell line stably transfected with GRP receptor (RIE/GRPR), suggesting the interaction between TGF-β signaling pathway and GRPR. The current study examined the biological responses of RIE/GRPR cells to TGF-β and BBS. Treatment with TGF-β1 (40 pM) and BBS (100 nM) together synergistically inhibited RIE/GRPR growth and induced apoptosis. Pretreatment with SB203580 (10 μM), a specific inhibitor of p38^MAPK, partially blocked the synergistic effect of TGF-β and BBS on apoptosis. In conclusion, BBS enhanced TGF-β growth inhibitory effect through apoptosis induction, which is at least partially mediated by p38^MAPK.     

A Shinella β-N-acetylglucosaminidase of glycoside hydrolase family 20 displays novel biochemical and molecular characteristics

β-N-Acetylglucosaminidases (GlcNAcases) are important for many biological functions and industrial applications. In this study, a glycoside hydrolase family 20 GlcNAcase from Shinella sp. JB10 was expressed in Escherichia coli BL21 (DE3). Compared to many GlcNAcases, the purified recombinant enzyme (rJB10Nag) exhibited a higher specificity activity (538.8 µmol min⁻¹ mg⁻¹) or V _max (1030.0 ± 82.1 µmol min⁻¹ mg⁻¹) toward p-nitrophenyl β-N-acetylglucosaminide and N,N′-diacetylchitobiose (specificity activity of 35.4 µmol min⁻¹ mg⁻¹) and a higher N-acetylglucosaminide tolerance (approximately 50% activity in 70.0 mM N-acetylglucosaminide). The degree of synergy on enzymatic degradation of chitin by a commercial chitinase and rJB10Nag was as high as 2.35. The enzyme was tolerant to most salts, especially 3.0–15.0% (w/v) NaCl and KCl. These biochemical characteristics make the JB10 GlcNAcase a candidate for use in many potential applications, including processing marine materials and the bioconversion of chitin waste. Furthermore, the enzyme has the highest proportions of alanine (16.5%), glycine (10.5%), and random coils (48.8%) with the lowest proportion of α-helices (24.9%) among experimentally characterized GH 20 GlcNAcases from other organisms.

     

A PGD Pregnancy Achieved by Embryo Copy Number Variation Sequencing with Confirmation by Non-Invasive Prenatal Diagnosis

Aneuploidy rates in embryos produced by assisted reproduc- tive technologies commonly exceed 50%, particularly in couples where the woman is of advanced maternal age or has experienced repeated implantation failure （Harper et al., 2012）. Pre-implantation genetic diagnosis （PGD） conducted at many fertility clinics worldwide is now a frontline treatment for infertile couples with a poor prognosis for pregnancy as well as couples where one partner is a carrier of a balanced translocation （Munne, 2012）.     

Disruption of the Homogentisate Solanesyltransferase Gene Results in Albino and Dwarf Phenotypes and Root,Trichome and Stomata Defects in Arabidopsis thaliana

Homogentisate solanesyltransferase (HST) plays an important role in plastoquinone (PQ) biosynthesis and acts as the electron acceptor in the carotenoids and abscisic acid (ABA) biosynthesis pathways. We isolated and identified a T-DNA insertion mutant of the HST gene that displayed the albino and dwarf phenotypes. PCR analyses and functional complementation also confirmed that the mutant phenotypes were caused by disruption of the HST gene. The mutants also had some developmental defects, including trichome development and stomata closure defects. Chloroplast development was also arrested and chlorophyll (Chl) was almost absent. Developmental defects in the chloroplasts were consistent with the SDS-PAGE result and the RNAi transgenic phenotype. Exogenous gibberellin (GA) could partially rescue the dwarf phenotype and the root development defects and exogenous ABA could rescue the stomata closure defects. Further analysis showed that ABA and GA levels were both very low in the pds2-1 mutants, which suggested that biosynthesis inhibition by GAs and ABA contributed to the pds2-1 mutants'' phenotypes. An early flowering phenotype was found in pds2-1 mutants, which showed that disruption of the HST gene promoted flowering by partially regulating plant hormones. RNA-sequencing showed that disruption of the HST gene resulted in expression changes to many of the genes involved in flowering time regulation and in the biosynthesis of PQ, Chl, GAs, ABA and carotenoids. These results suggest that HST is essential for chloroplast development, hormone biosynthesis, pigment accumulation and plant development.     

Characterization and mapping of a Prbs gene controlling spike development in Hordeum vulgare L.

The barley mutant, poly-row-and-branched spike (prbs) showed altered inflorescence morphology: complete conversion of the rudimentary lateral spikelets in two-rowed barley into fully developed fertile spikelets similar to the six-rowed phenotype, and additional spikelets in the middle of spike. Moreover, branched spikes emerged in progeny from a cross between the mutant and a six-rowed barley cultivar. Morphological observation of the development of immature spikes of the mutant and descendants with branched spikes showed that the Prbs gene is involved in spikelet development in the triple-mound stage. In mutant prbs, new meristems initiated at the flanks of lateral spikelets and middle spikelet meristems were converted to branch meristems, developing branched spikes. These observations suggested that the Prbs gene plays a crucial role in spikelet initiation and identity maintenance. The Prbs gene may be an important modifier in inflorescence differentiation from a panicle into a spike. The branched spikes emerging in hybrids from a cross between the mutant and six-rowed barley cultivar were not conferred by the gene vrs1 or Int-c, which decide spike morphology in six-rowed barley. These results imply that although six-row genes vrs1 and Int-c and prbs have similar effects on lateral spikelet development, they have different functions in branched spikes. The Prbs gene was mapped to chromosome 3H between SSR marker Bmag0023 and marker Cbic60 at a genetic distance of 3.3 and 5.4 centimorgans (cM), respectively.     

A global alfalfa diversity panel reveals genomic selection signatures in Chinese varieties and genomic associations with root development

Alfalfa (Medicago sativa L.) is an important forage crop worldwide. However, little is known about the effects of breeding status and different geographical populations on alfalfa improvement. Here, we sequenced 220 alfalfa core germplasms and determined that Chinese alfalfa cultivars form an independent group, as evidenced by comparisons of F_ST values between different subgroups, suggesting that geographical origin plays an important role in group differentiation. By tracing the influence of geographical regions on the genetic diversity of alfalfa varieties in China, we identified 350 common candidate genetic regions and 548 genes under selection. We also defined 165 loci associated with 24 important traits from genome-wide association studies. Of those, 17 genomic regions closely associated with a given phenotype were under selection, with the underlying haplotypes showing significant differences between subgroups of distinct geographical origins. Based on results from expression analysis and association mapping, we propose that 6-phosphogluconolactonase (MsPGL) and a gene encoding a protein with NHL domains (MsNHL) are critical candidate genes for root growth. In conclusion, our results provide valuable information for alfalfa improvement via molecular breeding.