Understanding the biologic role of N 6 -methyladenosine (m 6 A) RNA modifications in mRNA requires an understanding of when and where in the life of a pre-mRNA transcript the modifications are made. We found that HeLa cell chromatin-associated nascent pre-mRNA (CA-RNA) contains many unspliced introns and m 6 A in exons but very rarely in introns. The m 6 A methylation is essentially completed upon the release of mRNA into the nucleoplasm. Furthermore, the content and location of each m 6 A modification in steady-state cytoplasmic mRNA are largely indistinguishable from those in the newly synthesized CA-RNA or nucleoplasmic mRNA. This result suggests that quantitatively little methylation or demethylation occurs in cytoplasmic mRNA. In addition, only ∼10% of m 6 As in CA-RNA are within 50 nucleotides of 5 ′ or 3 ′ splice sites, and the vast majority of exons harboring m 6 A in wild-type mouse stem cells is spliced the same in cells lacking the major m 6 A methyltransferase Mettl3. Both HeLa and mouse embryonic stem cell mRNAs harboring m 6 As have shorter half-lives, and thousands of these mRNAs have increased half-lives (twofold or more) in Mettl3 knockout cells compared with wild type. In summary, m 6 A is added to exons before or soon after exon definition in nascent pre-mRNA, and while m 6 A is not required for most splicing, its addition in the nascent transcript is a determinant of cytoplasmic mRNA stability. Studying nascent RNA synthesis in cultured cells using very brief pulse labeling with radioactive nucleosides allowed a number of advances in understanding premRNA synthesis and processing in the era before rapid nucleic acid sequencing. Examples include polyA addition on pre-mRNA before completion of mRNA processing and cytoplasmic entry (Darnell et al. 1971;Edmonds et al. 1971) and locating the first known boundaries of eukaryotic polymerase II transcription units through studying labeled nascent adenovirus transcripts (Bachenheimer and Darnell 1975;Evans et al. 1977;Weber et al. 1977).These early experiments were joined by a cell fractionation technique originated by Wuarin and Schibler (1994) that uses a 1 M urea solution to liberate a "chromatin" fraction from nuclei. This fraction provides a stringent purification of growing nascent pre-mRNA chains, isolated as a chromatin-associated RNA fraction (referred to as CA-RNA), along with RNA polymerase II plus all nuclear DNA and associated histones. Using specific labeled DNA probes, Wuarin and Schibler (1994) demonstrated removal in liver cell nuclei of some, but not all, introns from two specific nascent pre-mRNAs: a transcription factor pre-mRNA involved in circadian rhythm and the HMG coA reductase pre-mRNA. Recently, Pandya-Jones and Black (2009) adapted this procedure to study the extent and order of intron removal in cultured human carcinoma cell nuclei, again showing that many, but not all, introns are removed in CA-RNA.

Summary Emerging variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are of clinical concern. In a cohort of 417 persons who had received the second dose of BNT162b2 (Pfizer–BioNTech) or mRNA-1273 (Moderna) vaccine at least 2 weeks previously, we identified 2 women with vaccine breakthrough infection. Despite evidence of vaccine efficacy in both women, symptoms of coronavirus disease 2019 developed, and they tested positive for SARS-CoV-2 by polymerase-chain-reaction testing. Viral sequencing revealed variants of likely clinical importance, including E484K in 1 woman and three mutations (T95I, del142–144, and D614G) in both. These observations indicate a potential risk of illness after successful vaccination and subsequent infection with variant virus, and they provide support for continued efforts to prevent and diagnose infection and to characterize variants in vaccinated persons. (Funded by the National Institutes of Health and others.)

Alcadeina (Alca) is an evolutionarily conserved type I membrane protein expressed in neurons. We show here that Alca strongly associates with kinesin light chain (K D E4-8 Â10 À9 M) through a novel tryptophan-and aspartic acid-containing sequence. Alca can induce kinesin-1 association with vesicles and functions as a novel cargo in axonal anterograde transport. JNK-interacting protein 1 (JIP1), an adaptor protein for kinesin-1, perturbs the transport of Alca, and the kinesin-1 motor complex dissociates from Alca-containing vesicles in a JIP1 concentration-dependent manner. Alca-containing vesicles were transported with a velocity different from that of amyloid b-protein precursor (APP)-containing vesicles, which are transported by the same kinesin-1 motor. Alca-and APP-containing vesicles comprised mostly separate populations in axons in vivo. Interactions of Alca with kinesin-1 blocked transport of APP-containing vesicles and increased b-amyloid generation. Inappropriate interactions of Alc-and APP-containing vesicles with kinesin-1 may promote aberrant APP metabolism in Alzheimer's disease.

The neuron specific RNA-binding proteins NOVA1 and NOVA2 are highly homologous alternative splicing regulators. NOVA proteins regulate at least 700 alternative splicing events in vivo, yet relatively little is known about the biologic consequences of NOVA action and in particular about functional differences between NOVA1 and NOVA2. Transcriptome-wide searches for isoform-specific functions, using NOVA1 and NOVA2 specific HITS-CLIP and RNA-seq data from mouse cortex lacking either NOVA isoform, reveals that NOVA2 uniquely regulates alternative splicing events of a series of axon guidance related genes during cortical development. Corresponding axonal pathfinding defects were specific to NOVA2 deficiency: Nova2-/- but not Nova1-/- mice had agenesis of the corpus callosum, and axonal outgrowth defects specific to ventral motoneuron axons and efferent innervation of the cochlea. Thus we have discovered that NOVA2 uniquely regulates alternative splicing of a coordinate set of transcripts encoding key components in cortical, brainstem and spinal axon guidance/outgrowth pathways during neural differentiation, with severe functional consequences in vivo.DOI: http://dx.doi.org/10.7554/eLife.14371.001