Structure and mechanism of the type I-G CRISPR effector.

Type I CRISPR systems are the most common CRISPR type found in bacteria. They use a multisubunit effector, guided by crRNA, to detect and bind dsDNA targets, forming an R-loop and recruiting the Cas3 enzyme to facilitate target DNA destruction, thus providing immunity against mobile genetic elements. Subtypes have been classified into families A-G, with type I-G being the least well understood. Here, we report the composition, structure and function of the type I-G Cascade CRISPR effector from Thioalkalivibrio sulfidiphilus, revealing key new molecular details. The unique Csb2 subunit processes pre-crRNA, remaining bound to the 3' end of the mature crRNA, and seven Cas7 subunits form the backbone of the effector. Cas3 associates stably with the effector complex via the Cas8g subunit and is important for target DNA recognition. Structural analysis by cryo-Electron Microscopy reveals a strikingly curved backbone conformation with Cas8g spanning the belly of the structure. These biochemical and structural insights shed new light on the diversity of type I systems and open the way to applications in genome engineering.

Repurposing the atypical type I-G CRISPR system for bacterial genome engineering.

The CRISPR-Cas system functions as a prokaryotic immune system and is highly diverse, with six major types and numerous sub-types. The most abundant are type I CRISPR systems, which utilize a multi-subunit effector, Cascade, and a CRISPR RNA (crRNA) to detect invading DNA species. Detection leads to DNA loading of the Cas3 helicase-nuclease, leading to long-range deletions in the targeted DNA, thus providing immunity against mobile genetic elements (MGE). Here, we focus on the type I-G system, a streamlined, 4-subunit complex with an atypical Cas3 enzyme. We demonstrate that Cas3 helicase activity is not essential for immunity against MGE in vivo and explore applications of the Thioalkalivibrio sulfidiphilus Cascade effector for genome engineering in Escherichia coli. Long-range, bidirectional deletions were observed when the lacZ gene was targeted. Deactivation of the Cas3 helicase activity dramatically altered the types of deletions observed, with small deletions flanked by direct repeats that are suggestive of microhomology mediated end joining. When donor DNA templates were present, both the wild-type and helicase-deficient systems promoted homology-directed repair (HDR), with the latter system providing improvements in editing efficiency, suggesting that a single nick in the target site may promote HDR in E. coli using the type I-G system. These findings open the way for further application of the type I-G CRISPR systems in genome engineering.

HBV HBx-Downregulated lncRNA LINC01010 Attenuates Cell Proliferation by Interacting with Vimentin.

Hepatitis B virus (HBV) infection is closely related to hepatocellular carcinoma (HCC) development. To investigate the mechanism of HBV causing HCC, we previously analyzed the transcription of the HBV-transgenic cell line HepG2-4D14 and parental HepG2 cells and identified a subset of long noncoding RNAs (lncRNAs) differentially expressed between them. In this study, we focus on lncRNA LINC01010, as it is significantly downregulated in HepG2-4D14 cells and in liver tissues of HCC patients, and positively correlated with survival. We found that HBV-encoded HBx can reduce the transcription of LINC01010. Functional analysis showed that the overexpression of LINC01010 inhibits proliferation, migration and invasion of HepG2 cells while the knockdown of LINC01010 promotes these processes. By taking the approach of RNA immunoprecipitation (RIP) and mass spectrometry, we identified that LINC01010 can interact with vimentin. Further studies demonstrated that LINC01010 negatively affects the vimentin network extension and causes more rapid subunit exchange and lower stability of vimentin filaments. In addition, LINC01010 can reduce the amount of insoluble vimentin within cells, which suggests that LINC01010 interfers with vimentin polymerization. These data indicate that LINC01010 can inhibit the assembly of vimentin filament. Thus, we revealed that HBV HBx-downregulated LINC01010, which suppresses cell proliferation and migration by negatively regulating the formation of vimentin filament. Taken together, LINC01010 is a potential tumor suppressor that may restrain HBV-related HCC development.

Lnc-ISG20 Inhibits Influenza A Virus Replication by Enhancing ISG20 Expression.

Long noncoding RNAs (lncRNAs) are involved in many aspects of cellular processes, including the antiviral immune response. To identify influenza A virus (IAV)-related lncRNAs, we performed RNA deep sequencing to compare the profiles of lncRNAs in A549 and HEK293T cells with or without IAV infection. We identified an IAV-upregulated lncRNA named lnc-ISG20 because it shares most of its sequence with ISG20. We found that lnc-ISG20 is an interferon-stimulated gene similar to ISG20. Overexpression of lnc-ISG20 inhibited IAV replication, while lnc-ISG20 knockdown favored viral replication, suggesting that lnc-ISG20 is inhibitory to IAV replication. Further study indicated that overexpression of lnc-ISG20 enhances ISG20 protein levels, while knockdown of lnc-ISG20 reduces ISG20 protein levels in A549 cells induced with poly(I·C) and Sendai virus. We demonstrated that lnc-ISG20 inhibits IAV replication in an ISG20-dependent manner. As lnc-ISG20 did not affect the mRNA level of ISG20, we postulated that lnc-ISG20 may function as endogenous RNA competing with ISG20 to enhance its translation. Indeed, we identified that microRNA 326 (miR-326) is a mutual microRNA for both ISG20 and lnc-ISG20 that targets the 3' untranslated region of ISG20 mRNA to inhibit its translation. We confirmed that lnc-ISG20 can bind miR-326, which in turn decreased the amount of miR-326 bound to ISG20 mRNA. In conclusion, we identified that the IAV-upregulated lnc-ISG20 is a novel interferon-stimulated gene that elicits its inhibitory effect on IAV replication by enhancing ISG20 expression. We demonstrated that lnc-ISG20 functions as a competitive endogenous RNA to bind miR-326 to reduce its inhibition of ISG20 translation. Our results revealed the mechanism by which lnc-ISG20 inhibits IAV replication.IMPORTANCE The replication of influenza A virus is regulated by host factors. However, the mechanisms by which lncRNAs regulate IAV infection are not well understood. We identified that lnc-ISG20 is upregulated during IAV infection and is also an interferon-stimulated gene. We demonstrated that lnc-ISG20 can enhance ISG20 expression, which in turn inhibits IAV replication. Our studies indicate that lnc-ISG20 functions as a competing endogenous RNA that binds miR-326 and reduces its inhibitory effect on ISG20. Taken together, our findings reveal the mechanistic details of lnc-ISG20 negatively regulating IAV replication. These findings indicate that lnc-ISG20 plays an important role during the host antiviral immune response.

LncRNA SAMD12-AS1 promotes cell proliferation and inhibits apoptosis by interacting with NPM1.

Chronic hepatitis B virus infection is a major risk factor for hepatocellular carcinoma. HBV infection affects lncRNA expression in infected cells, but the detailed mechanism and biological significance are not yet clear. In this study, we focused on exploring the function of the HBV-upregulated lncRNA SAMD12-AS1 in cell proliferation. We found that there is a higher level of SAMD12-AS1 expression in tumors than in adjacent nontumorous liver tissues. We showed that ectopic expression of SAMD12-AS1 promotes cell growth and blocks apoptosis, while knockdown of SAMD12-AS1 inhibits cell proliferation and enhances etoposide-induced apoptosis. Using RNA immunoprecipitation and mass spectrometry, we determined that SAMD12-AS1 interacts with NPM1 and confirmed that SAMD12-AS1(1-350) is required for the interaction with NPM1. As it is known that NPM1 interacts with the E3 ligase HDM2 and reduces HDM2-mediated p53 degradation, we examined whether SAMD12-AS1 can affect p53 stability. Overexpression of SAMD12-AS1 caused a reduction in p53 protein levels by shortening its half-life. Conversely, knockdown of SAMD12-AS1 prolonged the half-life of p53. We further demonstrated that SAMD12-AS1 increased the interaction of HDM2 and p53 and enhanced p53 ubiquitination. Our findings reveal that HBV-upregulated SAMD12-AS1 regulates cell proliferation and apoptosis via the NPM1-HDM2-p53 axis.