A sliding-bulge structure at the Dicer processing site of pre-miRNAs regulates alternative Dicer processing to generate 5'-isomiRs.

5'-isomiRs expand the repertoire of miRNA targets. However, how they are generated is not well understood. Previously, we showed that for some miRNAs in mammalian cells, Drosha cleaves at multiple sites to generate multiple pre-miRNAs that give rise to multiple 5'-isomiRs. Here, we showed that for some other miRNAs, 5'-isomiRs are generated by alternative Dicer processing. In addition, we showed that in miR-203, alternative Dicer processing is regulated by a conserved sliding-bulge structure at the Dicer processing site, which allows the pre-miRNA molecule to fold into two different structures that are processed differently by Dicer. So far no RNA motif that slides to change conformation and alter a protein-RNA interaction has been reported. Thus, our study revealed a novel RNA motif that regulates 5'-isomiR generation in some miRNAs. It might also contribute to regulating protein-RNA interactions in other biological processes, since it takes only one point mutation to generate the sliding bulge, and there are a large number of different RNAs in the cell.

Optimizing sgRNA structure to improve CRISPR-Cas9 knockout efficiency.

BACKGROUND: Single-guide RNA (sgRNA) is one of the two key components of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome-editing system. The current commonly used sgRNA structure has a shortened duplex compared with the native bacterial CRISPR RNA (crRNA)-transactivating crRNA (tracrRNA) duplex and contains a continuous sequence of thymines, which is the pause signal for RNA polymerase III and thus could potentially reduce transcription efficiency. RESULTS: Here, we systematically investigate the effect of these two elements on knockout efficiency and showed that modifying the sgRNA structure by extending the duplex length and mutating the fourth thymine of the continuous sequence of thymines to cytosine or guanine significantly, and sometimes dramatically, improves knockout efficiency in cells. In addition, the optimized sgRNA structure also significantly increases the efficiency of more challenging genome-editing procedures, such as gene deletion, which is important for inducing a loss of function in non-coding genes. CONCLUSIONS: By a systematic investigation of sgRNA structure we find that extending the duplex by approximately 5 bp combined with mutating the continuous sequence of thymines at position 4 to cytosine or guanine significantly increases gene knockout efficiency in CRISPR-Cas9-based genome editing experiments.

A CRISPR-Based Screen Identifies Genes Essential for West-Nile-Virus-Induced Cell Death.

West Nile virus (WNV) causes an acute neurological infection attended by massive neuronal cell death. However, the mechanism(s) behind the virus-induced cell death is poorly understood. Using a library containing 77,406 sgRNAs targeting 20,121 genes, we performed a genome-wide screen followed by a second screen with a sub-library. Among the genes identified, seven genes, EMC2, EMC3, SEL1L, DERL2, UBE2G2, UBE2J1, and HRD1, stood out as having the strongest phenotype, whose knockout conferred strong protection against WNV-induced cell death with two different WNV strains and in three cell lines. Interestingly, knockout of these genes did not block WNV replication. Thus, these appear to be essential genes that link WNV replication to downstream cell death pathway(s). In addition, the fact that all of these genes belong to the ER-associated protein degradation (ERAD) pathway suggests that this might be the primary driver of WNV-induced cell death.

The SPOROCYTELESS/NOZZLE gene is involved in controlling stamen identity in Arabidopsis.

The stamen, which consists of an anther and a filament, is the male reproductive organ in a flower. The specification of stamen identity in Arabidopsis (Arabidopsis thaliana) is controlled by a combination of the B genes APETALA3 (AP3) and PISTILLATA, the C gene AGAMOUS (AG), and the E genes SEPALLATA1 (SEP1) to SEP4. The "floral organ-building" gene SPOROCYTELESS/NOZZLE (SPL/NZZ) plays a central role in regulating anther cell differentiation. However, much less is known about how "floral organ identity" and floral organ-building genes interact to control floral organ development. In this study, we report that ectopic expression of SPL/NZZ not only affects flower development in the wild-type background but also leads to the transformation of petal-like organs into stamen-like organs in flowers of ap2-1, a weak ap2 mutant allele. Moreover, our loss-of-function analysis indicates that the spl/nzz mutant enhances the phenotype of the ag weak allele ag-4. Furthermore, ectopic expression and overexpression of SPL/NZZ altered expression of AG, SEP3, and AP2 in rosette leaves and flowers, while ectopic expression of SPL/NZZ resulted in ectopic expression of AG and SEP3 in the outer whorls of flowers. Our results indicate that the SPL/NZZ gene is engaged in controlling stamen identity via interacting with genes required for stamen identity in Arabidopsis.

Signaling of cell fate determination by the TPD1 small protein and EMS1 receptor kinase.

Sexual reproduction requires the specification of cells with distinct fates in plants and animals. The EMS1 (also known as EXS) leucine-rich repeat receptor-like kinase (LRR-RLK) and TPD1 small protein play key roles in regulating somatic and reproductive cell fate determination in Arabidopsis anthers. Here, we show that ectopic expression of TPD1 causes abnormal differentiation of somatic and reproductive cells in anthers. In addition, ectopic TPD1 activity requires functional EMS1. Yeast two-hybrid, pull-down, and coimmunoprecipitation analyses further demonstrate that TPD1 interacts with EMS1 in vitro and in vivo. Moreover, TPD1 induces EMS1 phosphorylation in planta. Thus, our results suggest that TPD1 serves as a ligand for the EMS1 receptor kinase to signal cell fate determination during plant sexual reproduction.

Expression of barley BAX Inhibitor-1 in carrots confers resistance to Botrytis cinerea.

SUMMARY BAX Inhibitor-1 (BI-1) is a protein that controls heterologous BAX-induced cell death, the hypersensitive reaction and abiotic stress-induced cell death in plants. When over-expressed in epidermal cells of barley, barley BI-1 induces susceptibility to the biotrophic fungal pathogen Blumeria graminis. When we expressed barley BI-1 in carrot susceptible to the necrotrophic fungus Botrytis cinerea, we obtained BI-1-mediated resistance to fungus-induced leaf cell death and less fungal spreading on the leaves. Barley BI-1 also mediated resistance to Chalara elegans in carrot roots. The results support the idea that cell death inhibition is an applicable approach to control cell-death-inducing pathogens in crop plants.

Changes in DNA base sequences in the mutant of Arabidopsis thaliana induced by low-energy N(+) implantation.

To reveal the mutation effect of low-energy ion implantation on Ambidopsis thaliana in vivo, T80II, a stable dwarf mutant, derived from the seeds irradiated by 30 keV N(+) with the dose of 80 X 10(15) ions/cm(2) was used for Random Amplified Polymorphic DNA (RAPD) and base sequence analysis. The results indicated that among total 397 RAPD bands observed, 52 bands in T80II were different from those of wild type showing a variation frequency 13.1%. In comparison with the sequences of A. thaliana in GenBank, the RAPD fragments in T80II were changed greatly in base sequences with an average rate of one base change per 16.8 bases. The types of base changes included base transition, transversion, deletion and insertion. Among the 275 base changes detected, single base substitutions (97.09%) occurred more frequently than base deletions and insertions (2.91%). And the frequency of base transitions (66.55%) was higher than that of base transversions (30.55%). Adenine, thymine, guanine or cytosine could be replaced by any of other three bases in cloned DNA fragments in T80II. It seems that thymine was more sensitive to the irradiation than other bases. The flanking sequences of the base changes in RAPD fragments in T80II were analyzed and the mutational "hotspot" induced by low-energy ion implantation was discussed.