Soluble immune checkpoints are dysregulated in COVID-19 and heavy alcohol users with HIV infection

Immune checkpoints (ICPs) consist of paired receptor-ligand molecules that exert inhibitory or stimulatory effects on immune defense, surveillance, regulation, and self-tolerance. ICPs exist in both membrane and soluble forms in vivo and in vitro. Imbalances between inhibitory and stimulatory membrane-bound ICPs (mICPs) in malignant cells and immune cells in the tumor immune microenvironment (TIME) have been well documented. Blockades of inhibitory mICPs have emerged as an immense breakthrough in cancer therapeutics. However, the origin, structure, production regulation, and biological significance of soluble ICPs (sICPs) in health and disease largely remains elusive. Soluble ICPs can be generated through either alternative mRNA splicing and secretion or protease-mediated shedding from mICPs. Since sICPs are found in the bloodstream, they likely form a circulating immune regulatory system. In fact, there is increasing evidence that sICPs exhibit biological functions including (1) regulation of antibacterial immunity, (2) interaction with their mICP compartments to positively or negatively regulate immune responses, and (3) competition with their mICP compartments for binding to the ICP blocking antibodies, thereby reducing the efficacy of ICP blockade therapies. Here, we summarize current data of sICPs in cancer and infectious diseases. We particularly focus on sICPs in COVID-19 and HIV infection as they are the two ongoing global pandemics and have created the world's most serious public health challenges. A storm of sICPs occurs in the peripheral circulation of COVID-19 patients and is associated with the severity of COVID-19. Similarly, sICPs are highly dysregulated in people living with HIV (PLHIV) and some sICPs remain dysregulated in PLHIV on antiretroviral therapy (ART), indicating these sICPs may serve as biomarkers of incomplete immune reconstitution in PLHIV on ART. We reveal that HIV infection in the setting of alcohol abuse exacerbates sICP dysregulation as PLHIV with heavy alcohol consumption have significantly elevated plasma levels of many sICPs. Thus, both stimulatory and inhibitory sICPs are present in the bloodstream of healthy people and their balance can be disrupted under pathophysiological conditions such as cancer, COVID-19, HIV infection, and alcohol abuse. There is an urgent need to study the role of sICPs in immune regulation in health and disease.

MicroRNA and Alternative mRNA Splicing Events in Cancer Drug Response/Resistance: Potent Therapeutic Targets

Cancer is a multifaceted disease that involves several molecular mechanisms including changes in gene expression. Two important processes altered in cancer that lead to changes in gene expression include altered microRNA (miRNA) expression and aberrant splicing events. MiRNAs are short non-coding RNAs that play a central role in regulating RNA silencing and gene expression. Alternative splicing increases the diversity of the proteome by producing several different spliced mRNAs from a single gene for translation. MiRNA expression and alternative splicing events are rigorously regulated processes. Dysregulation of miRNA and splicing events promote carcinogenesis and drug resistance in cancers including breast, cervical, prostate, colorectal, ovarian and leukemia. Alternative splicing may change the target mRNA 3′UTR binding site. This alteration can affect the produced protein and may ultimately affect the drug affinity of target proteins, eventually leading to drug resistance. Drug resistance can be caused by intrinsic and extrinsic factors. The interplay between miRNA and alternative splicing is largely due to splicing resulting in altered 3′UTR targeted binding of miRNAs. This can result in the altered targeting of these isoforms and altered drug targets and drug resistance. Furthermore, the increasing prevalence of cancer drug resistance poses a substantial challenge in the management of the disease. Henceforth, molecular alterations have become highly attractive drug targets to reverse the aberrant effects of miRNAs and splicing events that promote malignancy and drug resistance. While the miRNA–mRNA splicing interplay in cancer drug resistance remains largely to be elucidated, this review focuses on miRNA and alternative mRNA splicing (AS) events in breast, cervical, prostate, colorectal and ovarian cancer, as well as leukemia, and the role these events play in drug resistance. MiRNA induced cancer drug resistance; alternative mRNA splicing (AS) in cancer drug resistance; the interplay between AS and miRNA in chemoresistance will be discussed. Despite this great potential, the interplay between aberrant splicing events and miRNA is understudied but holds great potential in deciphering miRNA-mediated drug resistance.

MYC regulates a pan-cancer network of co-expressed oncogenic splicing factors

MYC is dysregulated in >50% of cancers, but direct targeting of MYC has been clinically unsuccessful. Targeting downstream MYC effector pathways represents an attractive alternative. MYC regulates alternative mRNA splicing, a hallmark of cancer, but the mechanistic links between MYC and the splicing machinery remain underexplored. Here, we identify a network of splicing factors (SFs) co-expressed as SF-modules in MYC-active breast tumors. Of these, one is a pan-cancer SF-module, correlating with MYC-activity across 33 tumor types. In mammary cell models, MYC activation leads to co-upregulation of pan-cancer module SFs and to changes in >4,000 splicing events. In breast cancer organoids, co-overexpression of the pan-cancer SF-module is sufficient to induce splicing events that are also MYC-regulated in patient tumors and to increase organoid size and invasiveness, while its knockdown decreases organoid size. Finally, we uncover a pan-cancer splicing signature of MYC activity which correlates with survival in multiple tumor types. Our findings provide insight into the mechanisms and function of MYC-regulated splicing and for the development of therapeutics for MYC-driven tumors.

Reduced RNA editing in the failing human heart mediates alternative circular RNA splicing

Background and purpose Post-transcriptional RNA editing is an important mechanism in the development of human diseases. RNA editing can affect RNA stability and alternative splicing. The aim of our study was to characterize RNA editing and its impact on alternative RNA splicing in the healthy and failing human heart. Methods and results Human heart samples of heart failure (HF) patients (n=20) and controls (n=10) were analyzed using RNA sequencing with subsequent analysis of RNA editing. We identified adenosine-to-inosine (A-to-I) editing as the major form of RNA editing in human hearts, being reduced in HF patients. Consistently, we found the editing enzyme ADAR2 reduced in HF patients. A-to-I RNA editing predominantly occurred in intronic regions of protein-coding genes, specifically in repetitive, primate-specific Alu elements which can affect RNA splicing. Indeed, we found 173 circular RNAs (circRNAs) regulated by alternative mRNA splicing in the failing heart. Loss of ADAR2 led to reduced RNA editing concomitant with an increase of circRNA, while overexpression reduced circRNA expression and enhanced RNA editing. Conclusion A-to-I editing is the major type of RNA editing in the human heart, being reduced in HF. We demonstrate a primate-specific alternative RNA splicing mechanism mediated by RNA editing in human hearts. The findings may be relevant to diseases with reduced RNA editing such as cancer, neurological and cardiac diseases. FUNDunding Acknowledgement Type of funding sources: None.

Splice-variant specific effects of a CACNA1H mutation associated with writer’s cramp

The CACNA1H gene encodes the α1 subunit of the low voltage-activated Cav3.2 T-type calcium channel, an important regulator of neuronal excitability. Alternative mRNA splicing can generate multiple channel variants with distinct biophysical properties and expression patterns. Two major splice variants, containing or lacking exon 26 (± 26) have been found in different human tissues. In this study, we report splice variant specific effects of a Cav3.2 mutation found in patients with autosomal dominant writer’s cramp, a specific type of focal dystonia. We had previously reported that the R481C missense mutation caused a gain of function effect when expressed in Cav3.2 (+ 26) by accelerating its recovery from inactivation. Here, we show that when the mutation is expressed in the short variant of the channel (− 26), we observe a significant increase in current density when compared to wild-type Cav3.2 (− 26) but the effect on the recovery from inactivation is lost. Our data add to growing evidence that the functional expression of calcium channel mutations depends on which splice variant is being examined.

Development of a novel tau propagation mouse model endogenously expressing 3 and 4 repeat tau isoforms

The phenomenon of "prion-like propagation" in which aggregates of abnormal amyloid-fibrilized protein propagate between neurons and spread pathology, is attracting attention as a new mechanism in neurodegenerative diseases. There is a strong correlation between the accumulation or spread of abnormal tau aggregates and the clinical symptoms of tauopathies. Microtubule-associated protein of tau contains a microtubule-binding domain which consists of 3-repeats or 4-repeats due to alternative mRNA splicing of transcripts for the Microtubule-associated protein of tau gene. Although a number of models for tau propagation have been reported, most utilize 4-repeat human tau transgenic mice or adult wild-type mice expressing only endogenous 4-repeat tau and these models have not been able to reproduce the pathology of Alzheimer's disease in which 3-repeat and 4-repeat tau accumulate simultaneously, or that of Pick’s disease in which only 3-repeat tau is aggregated. These deficiencies may reflect differences between human and rodent tau isoforms in the brain. To overcome this problem, we used genome editing techniques to generate mice that express an equal ratio of endogenous 3-repeat and 4-repeat tau, even after they become adults. We injected these mice with sarkosyl-insoluble fractions derived from the brains of human tauopathy patients such as those afflicted with Alzheimer’s disease (3- and 4-repeat tauopathy), corticobasal degeneration (4-repeat tauopathy) or Pick’s disease (3-repeat tauopathy). At 8-9 months following intracerebral injection of mice, histopathological and biochemical analyses revealed that the abnormal accumulation of tau was seed-dependent, with 3- and 4-repeat tau in Alzheimer’s disease-injected brains, 4-repeat tau only in corticobasal degeneration-injected brains, and 3-repeat tau only in Pick disease-injected brains, all of which contained isoforms related to those found in the injected seeds. The injected abnormal tau was seeded, and accumulated at the site of injection and at neural connections, predominantly within the same site. The abnormal tau newly accumulated was found to be endogenous in these mice and to have crossed the species barrier. Of particular importance, Pick’s body-like inclusions were observed in Pick’s disease-injected mice, and accumulations characteristic of Pick’s disease were reproduced, suggesting that we have developed the first model that recapitulates the pathology of Pick’s disease. These models are not only useful for elucidating the mechanism of propagation of tau pathology involving both 3- and 4-repeat-isoforms, but can also reproduce the pathology of tauopathies, which should lead to the discovery of new therapeutic agents.

Arg-tRNA synthetase links inflammatory metabolism to RNA splicing via nuclear condensates

SummaryCells respond to perturbations such as inflammation by sensing changes in metabolite levels. Especially prominent is arginine, which has long known connections to the inflammatory response. Here we show that depletion of arginine during inflammation decreased levels of arginyl-tRNA synthetase (ArgRS) in the nucleus. We found that nuclear ArgRS interacted with serine/arginine repetitive matrix protein 2 (SRRM2) in membrane-less, condensate-like, SRRM2-dependent nuclear speckles. This interaction impeded SRRM2 speckle trafficking and resulted in changes in alternative mRNA splicing. Splice site usage was regulated in opposite directions by ArgRS and SRRM2. These ArgRS- and SRRM2-dependent splicing changes cumulated in synthesis of different protein isoforms that altered cellular metabolism and peptide presentation to immune cells. Our findings delineate a novel mechanism whereby a tRNA synthetase responds to a metabolic change and modulates the splicing machinery via condensate trafficking for cellular responses to inflammatory injury.

Characterization and Dynamics of Intracellular Gene Transfer in Plastid Genomes of Viola (Violaceae) and Order Malpighiales

Functional gene transfer from organelles to the nucleus, known as intracellular gene transfer (IGT), is an ongoing process in flowering plants. The complete plastid genomes (plastomes) of two Ulleung island endemic violets, Viola ulleungdoensis and V. woosanensis, were characterized, revealing a lack of the plastid-encoded infA, rpl32, and rps16 genes. In addition, functional replacement of the three plastid-encoded genes in the nucleus was confirmed within the genus Viola and the order Malpighiales. Three strategies for the acquisition of a novel transit peptide for successful IGT were identified in the genus Viola. Nuclear INFA acquired a novel transit peptide with very low identity between these proteins, whereas the nuclear RPL32 gene acquired an existing transit peptide via fusion with the nuclear-encoded plastid-targeted SOD gene (Cu-Zn superoxide dismutase superfamily) as one exon, and translated both proteins in the cytosol using alternative mRNA splicing. Nuclear RPS16 contains an internal transit peptide without an N-terminal extension. Gene loss or pseudogenization of the plastid-borne rpl32 and rps16 loci was inferred to occur in the common ancestor of the genus Viola based on an infrageneric phylogenetic framework in Korea. Although infA was lost in the common ancestor of the order Malpighiales, the rpl32 and rps16 genes were lost multiple times independently within the order. Our current study sheds additional light on plastid genome composition and IGT mechanisms in the violet genus and in the order Malpighiales.

Plasma Membrane Ca2+ Pump PMCA4z Is More Active Than Splicing Variant PMCA4x

The plasma membrane Ca2+ pumps (PMCA) are P-ATPases that control Ca2+ signaling and homeostasis by transporting Ca2+ out of the eukaryotic cell. Humans have four genes that code for PMCA isoforms (PMCA1-4). A large diversity of PMCA isoforms is generated by alternative mRNA splicing at sites A and C. The different PMCA isoforms are expressed in a cell-type and developmental-specific manner and exhibit differential sensitivity to a great number of regulatory mechanisms. PMCA4 has two A splice variants, the forms “x” and “z”. While PMCA4x is ubiquitously expressed and relatively well-studied, PMCA4z is less characterized and its expression is restricted to some tissues such as the brain and heart muscle. PMCA4z lacks a stretch of 12 amino acids in the so-called A-M3 linker, a conformation-sensitive region of the molecule connecting the actuator domain (A) with the third transmembrane segment (M3). We expressed in yeast PMCA4 variants “x” and “z”, maintaining constant the most frequent splice variant “b” at the C-terminal end, and obtained purified preparations of both proteins. In the basal autoinhibited state, PMCA4zb showed a higher ATPase activity and a higher apparent Ca2+ affinity than PMCA4xb. Both isoforms were stimulated by calmodulin but PMCA4zb was more strongly activated by acidic lipids than PMCA4xb. The results indicate that a PMCA4 intrinsically more active and more responsive to acidic lipids is produced by the variant “z” of the splicing site A.