Summary 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 endoplasmic reticulum-associated protein degradation (ERAD) pathway suggests that this might be the primary driver of WNV-induced cell death.
Microprocessor [Drosha-DGCR8 (DiGeorge syndrome critical region gene 8) complex] processing of primary microRNA (pri-miRNA) is the critical first step in miRNA biogenesis, but how the Drosha cleavage site is determined has been unclear. Previous models proposed that the Drosha-DGCR8 complex measures either ∼22 nt from the upper stem-single-stranded RNA (ssRNA, terminal loop) junction or ∼11 nt from the lower stem-ssRNA junction to determine the cleavage site. Here, using miRNA-offset RNAs to determine the Drosha cleavage site, we show that the Microprocessor measures the distances from both the lower and upper stem-ssRNA junctions to determine the cleavage site in human cells, and optimal distances from both structures are critical to the precision of Drosha processing. If the distances are not optimal, Drosha tends to cleave at multiple sites, which can, in turn, generate multiple 5′ isomiRs. Thus, our results also reveal a mechanism of 5′ isomiR generation.moR | DcRNA | alternative Drosha processing I n mammals, the canonical pathway of miRNA biogenesis is initiated by the Drosha-DGCR8 (DiGeorge syndrome critical region gene 8) complex (the Microprocessor), which processes long primary miRNAs (pri-miRNAs) into ∼60-nt pre-miRNAs for further processing by Dicer into a duplex ∼22 nt long. One or both strands of the duplex are loaded into the RNA-induced silencing complex (RISC) to repress target gene expression (1, 2). Although the major players of miRNA biogenesis are mostly known, no model precisely predicts how the miRNA biogenesis machinery recognizes and processes a novel pri-miRNA or an miRNA-mimicking shRNA and which strand will finally be loaded into RISC. The missing details in miRNA biogenesis are major obstacles to rational design of miRNA-based shRNA or artificial miRNA (amiRNA) that generates predictable results, as this kind of amiRNA must be processed by the endogenous miRNA machinery to be functional. The lack of a precise model of miRNA biogenesis also makes it difficult to accurately predict novel miRNA genes in the genome. In fact, a recent study found that more than 150 miRNAs annotated in miRBase (of 564 miRNAs checked) were not miRNAs at all (3). Thus, there is still a need to improve our understanding of how the miRNA biogenesis machinery recognizes and processes pri-miRNA into mature miRNA.The secondary structure of a canonical pri-miRNA usually consists of four parts: a terminal loop, an upper stem that encompasses the mature miRNA duplex of approximately two helical turns, the lower stem, which is an approximate one-helical-turn extension of the miRNA duplex, and the basal segments, which are single-stranded flanking sequences (4, 5). In 2005, Zeng et al. proposed that a large terminal loop is required to guide Drosha cleavage to occur at ∼22 nt from the junction of the terminal loop single-stranded RNA (ssRNA) and upper stem (5). In addition, they showed that single-stranded flanking sequences around the miRNA hairpin structure are also required for efficient Drosha processing (6). Howe...
Pol III promoters such as U6 are commonly used to express small RNAs, including small interfering RNA, short hairpin RNA, and guide RNA, for the clustered regularly interspaced short palindromic repeats genome-editing system. However, whether the small RNAs were precisely expressed as desired has not been studied. Here, using deep sequencing to analyze small RNAs, we show that, for mouse U6 promoter, sequences immediately upstream of the putative initiation site, which is often modified to accommodate the restriction enzyme sites that enable easy cloning of small RNAs, are critical for precise transcription initiation. When the promoter is kept unmodified, transcription starts precisely from the first available A or G within the range of positions −1 to +2. In addition, we show that transcription from another commonly used pol III promoter, H1, starts at multiple sites, which results in variability at the 5′ end of the transcripts. Thus, inaccuracy of 5′ end of small RNA transcripts might be a common problem when using these promoters to express small RNAs based on currently believed concepts. Our study provides general guidelines for minimizing the variability of initiation, thereby enabling more accurate expression of small RNAs.
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