Selective Translational Repression of Truncated Proteins from Frameshift Mutation-Derived mRNAs in Tumors

Frameshift and nonsense mutations are common in tumors with microsatellite instability, and mRNAs from these mutated genes have premature termination codons (PTCs). Abnormal mRNAs containing PTCs are normally degraded by the nonsense-mediated mRNA decay (NMD) system. However, PTCs located within 50–55 nucleotides of the last exon–exon junction are not recognized by NMD (NMD-irrelevant), and some PTC-containing mRNAs can escape from the NMD system (NMD-escape). We investigated protein expression from NMD-irrelevant and NMD-escape PTC-containing mRNAs by Western blotting and transfection assays. We demonstrated that transfection of NMD-irrelevant PTC-containing genomic DNA of MARCKS generates truncated protein. In contrast, NMD-escape PTC-containing versions of hMSH3 and TGFBR2 generate normal levels of mRNA, but do not generate detectable levels of protein. Transfection of NMD-escape mutant TGFBR2 genomic DNA failed to generate expression of truncated proteins, whereas transfection of wild-type TGFBR2 genomic DNA or mutant PTC-containing TGFBR2 cDNA generated expression of wild-type protein and truncated protein, respectively. Our findings suggest a novel mechanism of gene expression regulation for PTC-containing mRNAs in which the deleterious transcripts are regulated either by NMD or translational repression

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

C-terminal substitution of HBV core proteins with those from DHBV reveals that arginine-rich 167RRRSQSPRR175 domain is critical for HBV replication.

To investigate the contributions of carboxyl-terminal nucleic acid binding domain of HBV core (C) protein for hepatitis B virus (HBV) replication, chimeric HBV C proteins were generated by substituting varying lengths of the carboxyl-terminus of duck hepatitis B virus (DHBV) C protein for the corresponding regions of HBV C protein. All chimeric C proteins formed core particles. A chimeric C protein with 221-262 amino acids of DHBV C protein, in place of 146-185 amino acids of the HBV C protein, supported HBV pregenomic RNA (pgRNA) encapsidation and DNA synthesis: 40% amino acid sequence identity or 45% homology in the nucleic-acid binding domain of HBV C protein was sufficient for pgRNA encapsidation and DNA synthesis, although we predominantly detected spliced DNA. A chimeric C protein with 221-241 and 251-262 amino acids of DHBV C, in place of HBV C 146-166 and 176-185 amino acids, respectively, could rescue full-length DNA synthesis. However, a reciprocal C chimera with 242-250 of DHBV C ((242)RAGSPLPRS(250)) introduced in place of 167-175 of HBV C ((167)RRRSQSPRR(175)) significantly decreased pgRNA encapsidation and DNA synthesis, and full-length DNA was not detected, demonstrating that the arginine-rich (167)RRRSQSPRR(175) domain may be critical for efficient viral replication. Five amino acids differing between viral species (underlined above) were tested for replication rescue; R169 and R175 were found to be important

Ca²⁺/Calmodulin-Dependent Protein Kinase II Inhibits Hepatitis B Virus Replication from cccDNA via AMPK Activation and AKT/mTOR Suppression

Ca2+/calmodulin-dependent protein kinase II (CaMKII), which is involved in the calcium signaling pathway, is an important regulator of cancer cell proliferation, motility, growth, and metastasis. The effects of CaMKII on hepatitis B virus (HBV) replication have never been evaluated. Here, we found that phosphorylated, active CaMKII is reduced during HBV replication. Similar to other members of the AMPK/AKT/mTOR signaling pathway associated with HBV replication, CaMKII, which is associated with this pathway, was found to be a novel regulator of HBV replication. Overexpression of CaMKII reduced the expression of covalently closed circular DNA (cccDNA), HBV RNAs, and replicative intermediate (RI) DNAs while activating AMPK and inhibiting the AKT/mTOR signaling pathway. Findings in HBx-deficient mutant-transfected HepG2 cells showed that the CaMKII-mediated AMPK/AKT/mTOR signaling pathway was independent of HBx. Moreover, AMPK overexpression reduced HBV cccDNA, RNAs, and RI DNAs through CaMKII activation. Although AMPK acts downstream of CaMKII, AMPK overexpression altered CaMKII phosphorylation, suggesting that CaMKII and AMPK form a positive feedback loop. These results demonstrate that HBV replication suppresses CaMKII activity, and that CaMKII upregulation suppresses HBV replication from cccDNA via AMPK and the AKT/mTOR signaling pathway. Thus, activation or overexpression of CaMKII may be a new therapeutic target against HBV infection

pgRNA encapsidation in core particles by additional C protein variants.

<p>(A) RPA to detect encapsidated pgRNA. To detect the pgRNA encapsidated by chimeric C protein variants, the C-deficient-RT-YMHA mutant and the C protein chimeras pHCP, pDCP, pHD192–262, pHD192–220, pHD221–262, pHD221–241, pHD242–262, pHDHD, or pHHDH were co-transfected into HuH7 cells. RPA (top panel) was performed as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041087#pone-0041087-g002" target="_blank">Figure 2D</a>. Core particle formation (second panel) and luciferase levels (bottom panel) were determined as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041087#pone-0041087-g001" target="_blank">Figure 1B</a>. Transfection experiments were repeated three times. (B) Relative levels of encapsidated pgRNA and core particle assembly by additional chimeric C protein variants. Relative levels of encapsidated pgRNA, core particles, and luciferase were measured with the Fujifilm Image Gauge V4.0 program. Relative levels of encapsidated pgRNA and core particle assembly were compared to normalized transfection efficiencies. The data represent the mean ± SD from three independent experiments.</p