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Until cytoplasmic recapping was discovered, decapping was thought to irreversibly destine an mRNA to degradation. Contradicting this idea, we readily observe mRNAs targeted by cytoplasmic capping in uncapped, yet stable forms. 5′ rapid amplification of cDNA ends (RACE) shows that nearly all uncapped ends correspond to capped analysis of gene expression tags and that the recapping of ZNF207 mRNA may be restricted to a single splice isoform. Here, a modified RACE approach detected uncapped 5′ RNA ends mapping to 46 mRNAs in cells expressing a dominant negative cytoplasmic capping enzyme and in normal cells. Eleven of 46 cloned mRNAs also contained splice isoform‐limiting sequences. Collectively, these data reinforce earlier work and suggest that alternative splicing may play a role in targeting transcripts for – and/or determining the position of – cytoplasmic capping. [ABSTRACT FROM AUTHOR]

Expression ofα1antichymotrypsin (ACT) is significantly activated by interleukin-1 (IL-1) in human astrocytes; however, it is barely affected by IL-1 in hepatocytes. This tissue-specific regulation depends upon an enhancer that contains both nuclear factorκB (NF-κB) and activating protein 1 (AP-1) elements, and is also observed for an NF-κB reporter but not for an AP-1 reporter. We found efficient activation of NF-κB binding in both cell types; however, this binding was persistent in glial cells and only transient in hepatocytes. IL-1-activated NF-κB complexes consisted of p65 and p50, with p65 transiently phosphorylated on serine 536 in glial cells whereas more persistently in hepatic cells. Overexpression of p65 or constitutively active IKKβ (inhibitor of NF-κB kinaseβ) resulted in an efficient activation of the ACT reporter in hepatic cells, indicating that a specific mechanism exists in these cells terminating IL-1 signaling. IL-1 effectively induced the degradation of inhibitor of NF-κBα (IkBα) and IkBε in both cell types but IkBβ was not affected. However, IkBα was resynthesized much more rapidly in hepatic cells in comparison to glial cells. In addition, the initial levels of IkBα were much lower in glial cells. We propose that the tissue-specific regulation of the ACT gene expression by IL-1 is determined by different efficiencies of IkBα resynthesis in glial and hepatic cells. [ABSTRACT FROM AUTHOR]

The COVID-19 mRNA vaccines demonstrated the power of mRNA medicines. Despite advancements in sequence design, evidence regarding the preferential use of synonymous codons on cellular stress and innate immune responses is lacking. To this end, we developed a proprietary codon optimality matrix to re-engineer the coding sequences of three luciferase reporters. We demonstrate that optimal mRNAs elicited dramatic increases in luciferase activities compared to non-optimal sequences. Notably, transfecting an optimal RNA affects the translation of other RNAs in the cell including control transcripts in dual luciferase assays. This held true in multiple cell lines and for an unrelated reporter. Further, non-optimal mRNAs preferentially activated innate immune pathways and the phosphorylation of the translation initiation factor eIF2α, a central event of the integrated stress response. Using nucleoside-modified or circular RNAs partially or fully abrogated these responses. Finally, we show that circularizing RNAs enhances both RNA lifespan and durability of protein expression. Our results show that RNA sequence, composition, and structure all govern RNA translatability. However, we also show that RNA sequences with poor codon optimality are immunogenic and induce cellular stress. Hence, RNA sequence engineering, chemical, and topological modifications must all be combined to elicit favorable therapeutic outcomes.

PCIF1 ( P hosphorylated C TD- I nteracting F actor 1 ) is the mRNA (2'-O-methyladenosine-N(6)-)-methyltransferase that catalyzes the formation of cap-adjacent N 6 ,2'-O-dimethyladenosine (m6Am) by methylating adenosines at the first transcribed position of capped mRNAs. While previous studies assumed that PCIF1 was nuclear, cell fractionation and immunofluorescence both show that a population of PCIF1 is localized to the cytoplasm. Further, PCIF1 redistributes to stress granules upon oxidative stress. Immunoprecipitation studies with stressed cells show that PCIF1 also physically interacts with G3BP and other stress granule components. In addition, PCIF1 behaves as a stress granule component as it disassociates from stress granules upon recovery from stress. Overexpressing full-length PCIF1 also inhibits stress granule formation, while knocking out PCIF1 slows stress granule disassembly. Next, our enhanced crosslinking and immunoprecipitation (eCLIP) data show that PCIF1 binds mRNAs in their coding sequences rather than cap-proximal regions. Further PCIF1's association with mRNAs increased upon NaAsO 2 stress. In contrast to eCLIP data, ChIP-Seq experiments show that PCIF1 is predominantly associated with transcription start sites rather than gene bodies, indicating that PCIF1's association with mature mRNA is not co-transcriptional. Collectively, our data suggest that PCIF1 has cytoplasmic RNA surveillance role(s) independent of transcription-associated cap-adjacent mRNA modification, particularly during the stress response.

Despite being under development for decades, RNA therapeutics have only recently emerged as viable drug platforms. The COVID-19 mRNA vaccines have demonstrated the promise and power of the platform technology. In response, novel RNA drugs are entering clinical trials at an accelerating rate. As the skin is the largest and most accessible organ, it has always been a preferred target for drug discovery. This holds true for RNA therapies as well, and multiple candidate RNA-based drugs are currently in development for an array of skin conditions. In this mini review, we catalog the RNA therapies currently in clinical trials for different dermatological diseases. We summarize the main types of RNA-related drugs and use examples of drugs currently in development to illustrate their key mechanism of action., Competing Interests: Conflict of Interest Dr. Kiss runs an externally funded laboratory (American Heart Association [20CDA35310329], and NIH [R35GM137819–03, −03S1, −03S2, −03S3], plus two subcontracts to NIH Contract #: 75N93019C00045) that is actively designing and testing different candidate RNA therapeutics. All authors anticipate seeking appropriate intellectual property protection for promising candidates that emerge from the lab’s work. Dr. Kiss and Dr. Bejar are named inventors on a recently filed patent for a novel RNA therapy platform. Dr. Kiss also has patents planned for several additional RNA therapeutics and an inducible stable HUVEC cell line. Dr. Bejar is named as a co-inventor on a patent filing for a candidate RNA therapeutic (no.17969496). Further, Dr. Kiss serves as an ad hoc consultant for multiple different for-profit companies and to the RNA Core at the Houston Methodist Research Institute. Dr. Kiss also reports private stock for BioLife Solutions Inc.