Biology subjects

Kiss, D. L.

Publications and source records attributed to Kiss, D. L..

6 recordsLinked to original sources

bioRxiv2026

Efficient permuted intron-exon RNA circularization system based on the LtrB group II Intron from Lactococcus lactis

Circular RNAs (circRNAs) have emerged as promising platforms for stable RNA expression and therapeutic applications, but methods for their generation remain limited. Here, we engineered the Lactococcus lactis LtrB group II intron into a permuted intron-exon (PIE) configuration to enable efficient circRNA formation (circLtrB). Among the tested split designs, LtrB domain IV cleavage yielded the highest circularization efficiency. Although LtrB encodes LtrA maturase required for efficient in vivo splicing, in vitro application of LtrA to the PIE constructs resulted in lower circularization efficiency than self-splicing conditions. We further examined circularization under distinct salt conditions and showed that NH4Cl- and KCl-based splicing buffers independently support high circularization efficiencies (>80%), while producing distinct splicing intermediates, indicating ion-specific mechanistic effects on circularization. Extending outer homology arms did not enhance circularization efficiency, demonstrating that efficient circRNA formation in the circLtrB platform is independent of extended exon pairing interactions. Using this system, we generated circRNAs across a range of coding sequence lengths, highlighting its payload flexibility. To optimize translation, we evaluated multiple internal ribosome entry sites (IRES), including Aichivirus, Crohivirus, CVB3, improved EMCV, HRV, and Salivirus, and found that the CVB3 IRES drove the highest translation efficiency. Linear NLuc showed faster decay kinetics, while circLtrB-NLuc exhibited more sustained expression over time. Transcriptome-wide RNA sequencing revealed comparable innate immune responses between circular and linear RNAs. Together, these results establish circLtrB as a versatile and efficient platform for circRNA production, providing mechanistic insight into group II intron-mediated circularization and expanding the toolkit for functional circRNA engineering.

molecular biology↗

bioRxiv2026

Kinetic Control of Nuclear-encoded Mitochondrial mRNA Localization and Local Translation

Most biological processes are dynamic, yet experimental methods predominantly rely on steady-state measurements to investigate their underlying mechanisms. RNA localization is a fundamental aspect of eukaryotic cell organization and is dynamically regulated by cells. While extensively studied in specialized cell types for a limited number of candidate RNAs, the general principles governing dynamic RNA localization at a transcriptome-wide scale remain largely unexplored. Existing transcriptome-wide studies provide only a static snapshot of RNAs residing in specific cellular locales, in part due to the limited availability of tools for probing cellular spatial organization at biologically relevant scales. Here, we leverage the high spatial (tens of nanometers) and temporal (minute) resolution of APEX-seq to quantitatively measure the dependence of RNA transport on molecular motors at a transcriptome-wide scale in living cells. We conducted these experiments in the context of the localization of mRNAs to the mitochondria, which are essential for cellular function. Our findings indicate that the majority of nuclear-encoded RNAs encoding mitochondrial proteins localize to the outer mitochondrial membrane (OMM) for local translation. We reveal a crucial role of retrograde dynein-based motor transport in RNA localization, demonstrating that its disruption severely impairs RNA targeting to the OMM. Time-resolved profiling of RNAs at the OMM revealed that localization is an active process, and even a brief disruption of transport for a few minutes results in a dramatic loss of localization. Moreover, we demonstrate that the translation efficiency (TE) of localized RNAs is a critical determinant of RNA localization in the context of motor-driven transport, as RNAs that delocalize following motor-transport perturbations exhibit lower TE. Using our temporal perturbation data, we also developed a spatiotemporal model that utilizes translation kinetics to capture key features of RNA localization dynamics at the OMM. Together, experiments and modeling suggest that the process of local translation at the OMM is kinetically controlled by the cell, and reveal an unappreciated mechanism by which active transport of RNAs enables cells to modulate their translation within minutes through RNA localization control. Our study demonstrates how simultaneously capturing the kinetics of hundreds of transcripts with minute resolution can uncover general principles of cellular and organelle organization. Together, these experiments and modeling reveal how active transport and translation jointly maintain the OMM-localized transcriptome. More broadly, they identify RNA localization to cellular membranes as a rapidly tunable mechanism for controlling local translation, even in non-polarized cells.

cell biology↗

bioRxiv2026

The RNase and RNA binding activities of selected RNase R truncations and mutations plus a detailed step by step protocol to purify recombinant RNase R

RNase R is a processive 3 to 5 exoribonuclease that degrades a broad array of linear RNA species while preserving RNA lariats and circular RNAs (circRNA). In recent years, this enzyme has become pivotal for the field of circRNA research, serving as a key step for circRNA enrichment, purification, and identification. Despite this growing importance, the effects of mutations and truncations in RNase R have been incompletely studied. We make several point mutations and assay their effects on the ability of RNase R to bind and/or degrade RNA substrates. Our data show that selected active site mutations have varying effects on RNA binding and degradation. Furthermore, the increasing interest in circRNA-based RNA therapeutic platforms highlights an urgent need for RNase R in RNA molecular biology labs. However, the substantial cost of commercial RNase R remains a bottleneck, particularly for large-scale studies or the development of circRNA-based technologies. In this protocol, we offer a solution to that problem, namely a more accessible and cost-effective means of purifying high-quality and low-cost RNase R. We provide a highly detailed yet simplified, high-yield protocol that produces recombinant RNase R from Escherichia coli. The method uses a single-step Ni-NTA affinity chromatography procedure without proteolytic tag removal and is optimized for entry-level FPLC systems such as the AKTA Start, ensuring that high-purity enzyme production does not require specialized, high-end instrumentation. A second key feature is the establishment of an optimized reaction framework, including specific buffer compositions and defined enzyme-to-substrate ratios for the purified RNase R. The protocol achieves functional equivalence to premium commercial RNase R, ensuring complete linear RNA digestion without compromising the integrity of circRNA. The combination of a simplified purification workflow and a robust reaction protocol provides an accessible, cost-effective, and reliable solution for any molecular biology laboratory requiring high volumes of RNase R. Key FeaturesO_LIRNase R mutations can block RNase activity, RNA binding or both C_LIO_LIThis protocol purifies [~]40 mg of active RNase R per liter of E. coli culture C_LIO_LIThe protocol avoids medium and high end FPLC systems C_LIO_LIRNase R expression constructs (WT and mutants) will be available on Addgene C_LIO_LIThe protocol includes an optimized reaction buffer to pair with this RNase R C_LIO_LIOptional endotoxin removal step is also included C_LI

molecular biology↗

bioRxiv2024

Codon optimality modulates cellular stress and innate immune responses triggered by exogenous RNAs

Despite advancements in RNA 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 new codon optimality formula to re-engineer the coding sequences of three luciferase reporters. We demonstrate that mRNAs enriched in optimal codons elicited dramatic increases in luciferase activities compared to less optimal sequences both in vitro and in vivo. Notably, transfecting low optimality test RNA suppresses the translation of co-transfected control mRNAs in dual reporter assays. Further, low optimality mRNAs activated innate immune pathways and the phosphorylation of the translation initiation factor eIF2, a central event of the integrated stress response. eIF2 phosphorylation was suppressed by a GCN2 inhibitor, but not by other eIF2 kinase inhibitors. Using nucleoside-modified or circular RNAs also fully or partially abrogated these responses. Finally, optimal, but not non-optimal, circular RNAs have enhanced RNA lifespan and duration of protein expression. Our results show that RNA sequence, composition, and structure all govern RNA translatability. Further, RNA sequences with poor codon optimality are immunogenic and induce cellular stress. Together, we show that RNA coding sequence design is a key consideration for both mRNA and circular RNA therapeutics.

molecular biology↗

bioRxiv2024

PCIF1 is partly cytoplasmic, dynamically localizes to stress granules and binds mRNA coding regions upon oxidative stress

PCIF1 (Phosphorylated CTD-Interacting Factor 1) is the mRNA (2-O-methyladenosine-N(6)-)-methyltransferase that catalyzes the formation of cap-adjacent N6,2-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 PCIF1s association with mRNAs increased upon NaAsO2 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 PCIF1s 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.

cell biology↗

bioRxiv2018

Truncated, uncapped mRNA 5’ ends targeted by cytoplasmic recapping cluster at CAGE tags and some transcripts are alternatively spliced

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 RACE shows that nearly all uncapped ends correspond to CAGE tags and that the recapping of ZNF207 mRNA may be restricted to a single splice isoform. A modified RACE approach detected uncapped 5 RNA ends mapping to 46 mRNAs in dominant negative cytoplasmic capping enzyme expressing and normal cells. 11 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.

molecular biology↗