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Splicing is a highly regulated process critical for proper pre-mRNA maturation and the maintenance of a healthy cellular environment. Splicing events are impacted by ongoing transcription, neighboring splicing events, and cis and trans regulatory factors on the respective pre-mRNA transcript. Within this complex regulatory environment, splicing kinetics have the potential to influence splicing outcomes but have historically been challenging to study in vivo. In this review, we highlight recent technological advancements that have enabled measurements of global splicing kinetics and of the variability of splicing kinetics at single introns. We demonstrate how identifying features that are correlated with splicing kinetics has increased our ability to form potential models for how splicing kinetics may be regulated in vivo., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Mitochondrial oxidative phosphorylation (OXPHOS) complexes are assembled from proteins encoded by both nuclear and mitochondrial DNA. These dual-origin enzymes pose a complex gene regulatory challenge for cells requiring coordinated gene expression across organelles. To identify genes involved in dual-origin protein complex synthesis, we performed fluorescence-activated cell-sorting-based genome-wide screens analysing mutant cells with unbalanced levels of mitochondrial- and nuclear-encoded subunits of Complex IV. We identified genes involved in OXPHOS biogenesis, including two uncharacterized genes: PREPL and NME6. We found that PREPL specifically impacts Complex IV biogenesis by acting at the intersection of mitochondrial lipid metabolism and protein synthesis, whereas NME6, an uncharacterized nucleoside diphosphate kinase, controls OXPHOS biogenesis through multiple mechanisms reliant on its NDPK domain. Firstly, NME6 forms a complex with RCC1L, which together perform nucleoside diphosphate kinase activity to maintain local mitochondrial pyrimidine triphosphate levels essential for mitochondrial RNA abundance. Secondly, NME6 modulates the activity of mitoribosome regulatory complexes, altering mitoribosome assembly and mitochondrial RNA pseudouridylation. Taken together, we propose that NME6 acts as a link between compartmentalized mitochondrial metabolites and mitochondrial gene expression., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Controlled release of promoter-proximal paused RNA polymerase II (RNA Pol II) is crucial for gene regulation. However, studying RNA Pol II pausing is challenging, as pause-release factors are almost all essential. In this study, we identified heterozygous loss-of-function mutations in SUPT5H, which encodes SPT5, in individuals with β-thalassemia. During erythropoiesis in healthy human cells, cell cycle genes were highly paused as cells transition from progenitors to precursors. When the pathogenic mutations were recapitulated by SUPT5H editing, RNA Pol II pause release was globally disrupted, and as cells began transitioning from progenitors to precursors, differentiation was delayed, accompanied by a transient lag in erythroid-specific gene expression and cell cycle kinetics. Despite this delay, cells terminally differentiate, and cell cycle phase distributions normalize. Therefore, hindering pause release perturbs proliferation and differentiation dynamics at a key transition during erythropoiesis, identifying a role for RNA Pol II pausing in temporally coordinating the cell cycle and erythroid differentiation., Competing Interests: Declaration of interests V.G.S. serves as an advisor to and/or has equity in Branch Biosciences, Ensoma, Novartis, Forma, Sana Biotechnology, and Cellarity, all unrelated to the present work. J.C.U. is an employee of Illumina, Inc., unrelated to the present work. R.I. is a founder, board member, and shareholder of Cellforma, unrelated to the present work., (Copyright © 2023 Elsevier Inc. All rights reserved.)

During maturation, eukaryotic precursor RNAs undergo processing events including intron splicing, 3'-end cleavage, and polyadenylation. Here we describe nanopore analysis of co-transcriptional processing (nano-COP), a method for probing the timing and patterns of RNA processing. An extension of native elongating transcript sequencing, which quantifies transcription genome-wide through short-read sequencing of nascent RNA 3' ends, nano-COP uses long-read nascent RNA sequencing to observe global patterns of RNA processing. First, nascent RNA is stringently purified through a combination of 4-thiouridine metabolic labeling and cellular fractionation. In contrast to cDNA or short-read-based approaches relying on reverse transcription or amplification, the sample is sequenced directly through nanopores to reveal the native context of nascent RNA. nano-COP identifies both active transcription sites and splice isoforms of single RNA molecules during synthesis, providing insight into patterns of intron removal and the physical coupling between transcription and splicing. The nano-COP protocol yields data within 3 d.

The aggregation and deposition of tau is a hallmark of a class of neurodegenerative diseases called tauopathies. Despite intensive study, cellular and molecular factors that trigger tau aggregation are not well understood. Here, we provide evidence for two mechanisms relevant to the initiation of tau aggregation in the presence of cytoplasmic polyphosphates (polyP): changes in the conformational ensemble of monomer tau and noncovalent cross-linking of multiple tau monomers. We identified conformational changes throughout full-length tau, most notably diminishment of long-range interactions between the termini coupled with compaction of the microtubule binding and proline- rich regions. We found that while the proline-rich and microtubule binding regions both contain polyP binding sites, the proline-rich region is a requisite for compaction of the microtubule binding region upon binding. Additionally, both the magnitude of the conformational change and the aggregation of tau are dependent on the chain length of the polyP polymer. Longer polyP chains are more effective at intermolecular, noncovalent cross-linking of tau. These observations provide an understanding of the initial steps of tau aggregation through interaction with a physiologically relevant aggregation inducer., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)