Rapid and inexpensive bedside diagnosis of RAN binding protein 2-associated acute necrotizing encephalopathy.

Acute necrotizing encephalopathy 1 (ANE1) is a very rare disorder associated with a dominant heterozygous mutation in the RANBP2 (RAN binding protein 2) gene. ANE1 is frequently triggered by a febrile infection and characterized by serious and irreversible neurological damage. Although only a few hundred cases have been reported, mutations in RANBP2 are only partially penetrant and can occur de novo, suggesting that their frequency may be higher in some populations. Genetic diagnosis is a lengthy process, potentially delaying definitive diagnosis. We therefore developed a rapid bedside qPCR-based tool for early diagnosis and screening of ANE1 mutations. Primers were designed to specifically assess RANBP2 and not RGPD (RANBP2 and GCC2 protein domains) and discriminate between wild-type or mutant RANBP2. Nasal epithelial cells were obtained from two individuals with known RANBP2 mutations and two healthy control individuals. RANBP2-specific reverse transcription followed by allele-specific primer qPCR amplification confirmed the specific detection of heterozygously expressed mutant RANBP2 in the ANE1 samples. This study demonstrates that allele-specific qPCR can be used as a rapid and inexpensive diagnostic tool for ANE1 using preexisting equipment at local hospitals. It can also be used to screen non-hospitalized family members and at risk-population to better establish the frequency of non-ANE-associated RANBP2 mutations, as well as possible tissue-dependent expression patterns. Systematic review registration: The protocol was registered in the international prospective register of systematic reviews (PROSPERO- CRD42023443257).

TAG-RNAi overcomes off-target effects in cancer models.

RNA interference offers therapeutic opportunities for the clinical targeting of otherwise undruggable oncogenes. However RNAi can have off-target effects that considerably increase treatment risks. To manage these side effects and allow an easy subtraction of their activity in healthy tissues, we present here the TAG-RNAi approach where cells that are not designated targets do not have the mRNA tag. Using TAG-RNAi we first established the off-target signatures of three different siRNAs specific to the Cyclin D1 oncogene by RNA-sequencing of cultured cancer cells expressing a FLAG-HA-tagged-Cyclin D1. Then, by symmetrical allografts of tagged-cancer cells and untagged controls on the left and right flanks of model mice, we demonstrate that TAG-RNAi is a reliable approach to study the functional impact of any oncogene without off-target bias. Finally we show, as examples, that mutation-specific TAG-RNAi can be applied to downregulate two oncogenic mutants, KRAS-G12V or BRAF-V600E, while sparing the expression of the wild-type proteins. TAG-RNAi will thus avoid the traditional off-target limitations of RNAi in future experimental approaches.

Hepatitis B virus X protein identifies the Smc5/6 complex as a host restriction factor.

Chronic hepatitis B virus infection is a leading cause of cirrhosis and liver cancer. Hepatitis B virus encodes the regulatory HBx protein whose primary role is to promote transcription of the viral genome, which persists as an extrachromosomal DNA circle in infected cells. HBx accomplishes this task by an unusual mechanism, enhancing transcription only from extrachromosomal DNA templates. Here we show that HBx achieves this by hijacking the cellular DDB1-containing E3 ubiquitin ligase to target the 'structural maintenance of chromosomes' (Smc) complex Smc5/6 for degradation. Blocking this event inhibits the stimulatory effect of HBx both on extrachromosomal reporter genes and on hepatitis B virus transcription. Conversely, silencing the Smc5/6 complex enhances extrachromosomal reporter gene transcription in the absence of HBx, restores replication of an HBx-deficient hepatitis B virus, and rescues wild-type hepatitis B virus in a DDB1-knockdown background. The Smc5/6 complex associates with extrachromosomal reporters and the hepatitis B virus genome, suggesting a direct mechanism of transcriptional inhibition. These results uncover a novel role for the Smc5/6 complex as a restriction factor selectively blocking extrachromosomal DNA transcription. By destroying this complex, HBx relieves the inhibition to allow productive hepatitis B virus gene expression.

Hepatitis B virus X protein stimulates gene expression selectively from extrachromosomal DNA templates.

UNLABELLED: Chronic hepatitis B virus (HBV) infection is a major risk factor for liver cancer development. HBV encodes the hepatitis B virus X (HBx) protein that promotes transcription of the viral episomal DNA genome by the host cell RNA polymerase II. Here we provide evidence that HBx accomplishes this task by a conserved and unusual mechanism. Thus, HBx strongly stimulates expression of transiently transfected reporter constructs, regardless of the enhancer and promoter sequences. This activity invariably requires HBx binding to the cellular UV-damaged DDB1 E3 ubiquitin ligase, suggesting a common mechanism. Unexpectedly, none of the reporters tested is stimulated by HBx when integrated into the chromosome, despite remaining responsive to their cognate activators. Likewise, HBx promotes gene expression from the natural HBV episomal template but not from a chromosomally integrated HBV construct. The same was observed with the HBx protein of woodchuck HBV. HBx does not affect nuclear plasmid copy number and functions independently of CpG dinucleotide methylation. CONCLUSION: We propose that HBx supports HBV gene expression by a conserved mechanism that acts specifically on episomal DNA templates independently of the nature of the cis-regulatory sequences. Because of its uncommon property and key role in viral transcription, HBx represents an attractive target for new antiviral therapies.

Essential role for the interaction between hnRNP H/F and a G quadruplex in maintaining p53 pre-mRNA 3'-end processing and function during DNA damage.

Following DNA damage, mRNA 3'-end formation is inhibited, contributing to repression of mRNA synthesis. Here we investigated how DNA-damaged cells accomplish p53 mRNA 3'-end formation when normal mechanisms of pre-mRNA 3'-end processing regulation are inhibited. The underlying mechanism involves the interaction between a G-quadruplex structure located downstream from the p53 cleavage site and hnRNP H/F. Importantly, this interaction is critical for p53 expression and contributes to p53-mediated apoptosis. Our results uncover the existence of a specific rescue mechanism of 3'-end processing regulation allowing stress-induced p53 accumulation and function in apoptosis.

A physical and functional link between splicing factors promotes pre-mRNA 3' end processing.

Polypyrimidine tract-binding protein (PTB) is a splicing regulator that also plays a positive role in pre-mRNA 3' end processing when bound upstream of the polyadenylation signal (pA signal). Here, we address the mechanism of PTB stimulatory function in mRNA 3' end formation. We identify PTB as the protein factor whose binding to the human beta-globin (HBB) 3' UTR is abrogated by a 3' end processing-inactivating mutation. We show that PTB promotes both in vitro 3' end cleavage and polyadenylation and recruits directly the splicing factor hnRNP H to G-rich sequences associated with several pA signals. Increased binding of hnRNP H results in stimulation of polyadenylation through a direct interaction with poly(A) polymerase. Therefore, our results provide evidence of a concerted regulation of pA signal recognition by splicing factors bound to auxiliary polyadenylation sequence elements.