Insights from structural studies of the Cardiovirus 2A protein

Cardioviruses are single-stranded RNA viruses of the family Picornaviridae. In addition to being the first example of internal ribosome entry site utilization, cardioviruses also employ a series of alternative translation strategies, such as Stop-Go translation and programmed ribosome frameshifting. Here, we focus on cardiovirus 2A protein, which is not only a primary virulence factor, but also exerts crucial regulatory functions during translation, including activation of viral ribosome frameshifting and inhibition of host cap-dependent translation. Only recently, biochemical and structural studies have allowed us to close the gaps in our knowledge of how cardiovirus 2A is able to act in diverse translation-related processes as a novel RNA-binding protein. This review will summarize these findings, which ultimately may lead to the discovery of other RNA-mediated gene expression strategies across a broad range of RNA viruses.

G-Quadruplex Regulation of VEGFA mRNA Translation by RBM4

In eukaryotes, mRNAs translation is mainly mediated in a cap-dependent or cap-independent manner. The latter is primarily initiated at the internal ribosome entry site (IRES) in the 5′-UTR of mRNAs. It has been reported that the G-quadruplex structure (G4) in the IRES elements could regulate the IRES activity. We previously confirmed RBM4 (also known as LARK) as a G4-binding protein in human. In this study, to investigate whether RBM4 is involved in the regulation of the IRES activity by binding with the G4 structure within the IRES element, the IRES-A element in the 5′-UTR of vascular endothelial growth factor A (VEGFA) was constructed into a dicistronic reporter vector, psiCHECK2, and the effect of RBM4 on the IRES activity was tested in 293T cells. The results showed that the IRES insertion significantly increased the FLuc expression activity, indicating that this G4-containing IRES was active in 293T cells. When the G4 structure in the IRES was disrupted by base mutation, the IRES activity was significantly decreased. The IRES activity was notably increased when the cells were treated with G4 stabilizer PDS. EMSA results showed that RBM4 specifically bound the G4 structure in the IRES element. The knockdown of RBM4 substantially reduced the IRES activity, whereas over-expressing RBM4 increased the IRES activity. Taking all results together, we demonstrated that RBM4 promoted the mRNA translation of VEGFA gene by binding to the G4 structure in the IRES.

Expanding uncapped translation and emerging function of circular RNA in carcinomas and noncarcinomas

Circular RNAs (circRNAs) are classified as noncoding RNAs because they are devoid of a 5’ end cap and a 3’ end poly (A) tail necessary for cap-dependent translation. However, increasing numbers of translated circRNAs identified through high-throughput RNA sequencing overlapping with polysome profiling indicate that this rule is being broken. CircRNAs can be translated in cap-independent mechanism, including IRES (internal ribosome entry site)-initiated pattern, MIRES (m6A internal ribosome entry site) -initiated patterns, and rolling translation mechanism (RCA). CircRNA-encoded proteins harbour diverse functions similar to or different from host proteins. In addition, they are linked to the modulation of human disease including carcinomas and noncarcinomas. CircRNA-related translatomics and proteomics have attracted increasing attention. This review discusses the progress and exclusive characteristics of circRNA translation and highlights the latest mechanisms and regulation of circRNA translatomics. Furthermore, we summarize the extensive functions and mechanisms of circRNA-derived proteins in human diseases, which contribute to a better understanding of intricate noncanonical circRNA translatomics and proteomics and their therapeutic potential in human diseases.

First Experience With The GoBack-Catheter For Successful Crossing of Complex Chronic Total Occlusions in Lower Limb Arteries

Purpose: To evaluate the use of the GoBack-catheter (Upstream Peripheral Technologies) in complex revascularizations in lower limb arteries. Materials and Methods: In this retrospective single-center study, the results of the first 100 consecutive patients including 101 limb-revascularizations, performed between May 2018 and July 2020 with the study device, were analyzed. In all cases, guidewire-crossing failed, and all lesions were chronic total occlusions (CTO), either de novo, reocclusions, or in-stent reocclusions. Successful crossing was defined as passing the CTO using the study device. Patency at discharge and after 30 days was defined as less than 50% restenosis on duplex sonography, without target lesion revascularization. Results: Median lesion length was 24 cm and 38 patients (37.6%) had a calcium grading according to the peripheral arterial calcium scoring system (PACSS) of 4 or 5. In 20.8% of patients, an occluded stent was treated. CTOs involved the femoropopliteal segment in 91.1%, iliac arteries in 5.9%, and tibial arteries in 7.9%. The GoBack-catheter was employed for entering into or crossing through parts or the full length of a CTO or an occluded stent as well as for re-entering into the true lumen after subintimal crossing. The device was used via contralateral and ipsilateral antegrade as well as retrograde access with an overall technical success rate of 92.1%. In 3 patients minor bleeding occurred at the crossing or re-entry site, which were managed conservatively. Thirty-day adverse limb events comprised minor amputations in 4 patients (4.0%), 1 major amputation (1.0%), and reocclusions in 7 limbs (6.9%). Conclusion: The new GoBack-catheter offers versatile endovascular applicability for complex CTO recanalization in a broad range of peripheral vascular interventions with a high technical success and low complication rate.

Phenotypic Spectrum of Dystrophinopathy Due to Duchenne Muscular Dystrophy Exon 2 Duplications

Background and Objectives:To describe the phenotypic spectrum of dystrophinopathy in a large cohort of individuals with DMD exon 2 duplications (Dup2), whom may be particularly amenable to therapies directed at restoring expression of either full-length dystrophin, or nearly full-length dystrophin through utilization of the DMD exon 5 internal ribosome entry site (IRES).Methods:In this retrospective observational study, we analyzed data from large genotype-phenotype databases (the United Dystrophinopathy Project [UDP] and the Italian DMD network) and classified subjects into Duchenne (DMD), intermediate (IMD), or Becker (BMD) phenotypes. Log-rank tests for time-to event variables were used to compare age at loss of ambulation (LOA) in Dup2 subjects versus non-Dup2 controls in the UDP database, and for comparisons between steroid-treated vs. steroid-naive Dup2 subjects.Results:Among 66 Dup2 subjects (UDP=40, Italy=26), 61% percent were classified as DMD, 9% as IMD, and 30% as BMD. Median age at last observation was 15.4 years [IQR 8.79-26.0], and 75% had been on corticosteroids for at least 6 months. Age at LOA differed significantly between Dup2 DMD subjects and historical non-Dup2 DMD controls (p<0.001). Valid spirometry was limited but suggested a delay in the typical age-related decline in forced vital capacity, and 24 of 55 subjects with adequate cardiac data had cardiomyopathy.Discussion:Some Dup2 patients display a milder disease course than non-Dup2 DMD controls, and prolonged ambulation with corticosteroids suggests the potential of IRES activation as a molecular mechanism. As Dup2-targeted therapies reach clinical applications, this information is critical to aid in the interpretation of the efficacy of new treatments.

Distal Radial Artery Access for Coronary and Peripheral Procedures: A Multicenter Experience

Introduction: Distal radial access (dRA) has recently gained global popularity as an alternative access route for vascular procedures. Among the benefits of dRA are the low risk of entry site bleeding complications, the low rate of radial artery occlusion, and improved patient and operator comfort. The aim of this large multicenter registry was to demonstrate the feasibility and safety of dRA in a wide variety of routine procedures in the catheterization laboratory, ranging from coronary angiography and percutaneous coronary intervention to peripheral procedures. Methods: The study comprised 1240 patients who underwent coronary angiography, PCI or noncoronary procedures through dRA in two Hungarian centers from January 2019 to April 2021. Baseline patient characteristics, number and duration of arterial punctures, procedural success rate, crossover rate, postoperative compression time, complications, hospitalization duration, and different learning curves were analyzed. Results: The average patient age was 66.4 years, with 66.8% of patients being male. The majority of patients (74.04%) underwent a coronary procedure, whereas 25.96% were involved in noncoronary interventions. dRA was successfully punctured in 97% of all patients, in all cases with ultrasound guidance. Access site crossover was performed in 2.58% of the patients, mainly via the contralateral dRA. After experiencing 150 cases, the dRA success rate plateaued at >96%. Our dedicated dRA step-by step protocol resulted in high open radial artery (RA) rates: distal and proximal RA pulses were palpable in 99.68% of all patients at hospital discharge. The rate of minor vascular complications was low (1.5%). A threshold of 50 cases was sufficient for already skilled radial operators to establish a reliable procedural method of dRA access. Conclusion: The implementation of distal radial artery access in the everyday routine of a catheterization laboratory for coronary and noncoronary interventions is feasible and safe with an acceptable learning curve.

Molecular architecture of 40S initiation complexes on the Hepatitis C virus IRES: from ribosomal attachment to eIF5B-mediated reorientation of initiator tRNA

Hepatitis C virus mRNA contains an internal ribosome entry site (IRES) that mediates end-independent translation initiation, requiring a subset of eukaryotic initiation factors (eIFs). Direct binding of the IRES to the 40S subunit places the initiation codon into the P site, where it base-pairs with eIF2-bound Met-tRNAiMet forming a 48S initiation complex. Then, eIF5 and eIF5B mediate subunit joining. Initiation can also proceed without eIF2, in which case Met-tRNAiMet is recruited directly by eIF5B. Here, we present cryo-EM structures of IRES initiation complexes at resolutions up to 3.5 Å that cover all major stages from initial ribosomal association, through eIF2-containing 48S initiation complexes, to eIF5B-containing complexes immediately prior to subunit joining. These structures provide insights into the dynamic network of 40S/IRES contacts, highlight the role for IRES domain II, and reveal conformational changes that occur during the transition from eIF2- to eIF5B-containing 48S complexes that prepare them for subunit joining.

HSV-1 and influenza infection induce linear and circular splicing of the long NEAT1 isoform

The herpes simplex virus 1 (HSV-1) virion host shut-off (vhs) protein cleaves both cellular and viral mRNAs but not circular RNAs (circRNAs) without an internal ribosome entry site. Here, we show that vhs activity leads to an accumulation of circRNAs relative to linear mRNAs during HSV-1 infection. Strikingly, we found that circular splicing of the long isoform (NEAT1_2) of the nuclear paraspeckle assembly transcript 1 (NEAT1) was massively induced during HSV-1 infection in a vhs-independent manner while NEAT1_2 was still bound to the chromatin. This was associated with induction of linear splicing of NEAT1_2 both within and downstream of the circRNA. NEAT1_2 splicing was absent in uninfected cells but can be induced by ectopic co-expression of the HSV-1 immediate-early proteins ICP22 and ICP27. Interestingly, NEAT1_2 circular and linear splicing was also up-regulated in influenza infection but absent in stress conditions, which disrupt transcription termination similar to but not in the same manner as HSV-1 and influenza infection. Finally, large-scale analysis of published RNA-seq data uncovered induction of NEAT1_2 splicing in cancer cells upon inhibition or knockdown of cyclin-dependent kinase 7 (CDK7) or the MED1 subunit of the Mediator complex phosphorylated by CDK7. Interestingly, CDK7 inhibition also disrupted transcription termination, highlighting a possible link between disruption of transcription termination and NEAT1_2 splicing.