Explore the vast range of titles available.

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. In this extension to their NET-seq protocol, the authors combine isolation of 4sU-labeled chromatin-associated nascent RNA with long-read direct RNA sequencing on nanopores to profile the kinetics and patterns of co-transcriptional RNA processing.

Background Small nucleolar RNAs (snoRNAs) are abundant noncoding RNAs best known for their involvement in ribosomal RNA maturation. In mammals, most expressed snoRNAs are embedded in introns of longer genes and produced through transcription and splicing of their host. Intronic snoRNAs were long viewed as inert passengers with little effect on host expression. However, a recent study reported a snoRNA influencing the splicing and ultimate output of its host gene. Overall, the general contribution of intronic snoRNAs to host expression remains unclear. Results Computational analysis of large-scale human RNA-RNA interaction datasets indicates that 30% of detected snoRNAs interact with their host transcripts. Many snoRNA-host duplexes are located near alternatively spliced exons and display high sequence conservation suggesting a possible role in splicing regulation. The study of the model SNORD2-EIF4A2 duplex indicates that the snoRNA interaction with the host intronic sequence conceals the branch point leading to decreased inclusion of the adjacent alternative exon. Extended SNORD2 sequence containing the interacting intronic region accumulates in sequencing datasets in a cell-type-specific manner. Antisense oligonucleotides and mutations that disrupt the formation of the snoRNA-intron structure promote the splicing of the alternative exon, shifting the EIF4A2 transcript ratio away from nonsense-mediated decay. Conclusions Many snoRNAs form RNA duplexes near alternative exons of their host transcripts, placing them in optimal positions to control host output as shown for the SNORD2-EIF4A2 model system. Overall, our study supports a more widespread role for intronic snoRNAs in the regulation of their host transcript maturation.

Background Small nucleolar RNAs (snoRNAs) are non-coding RNAs that function in ribosome and spliceosome biogenesis, primarily by guiding modifying enzymes to specific sites on ribosomal RNA (rRNA) and spliceosomal RNA (snRNA). However, many orphan snoRNAs remain uncharacterized, with unidentified or unvalidated targets, and studies on additional snoRNA-associated proteins are limited. Results We adapted an enhanced chimeric eCLIP approach to comprehensively profile snoRNA-target RNA interactions using both core and accessory snoRNA-binding proteins as baits. Using core snoRNA-binding proteins, we confirmed most annotated snoRNA-rRNA and snoRNA-snRNA interactions in mouse and human cell lines and called novel, high-confidence interactions for orphan snoRNAs. While some of these interactions result in chemical modification, others may have modification-independent functions. We showed that snoRNA ribonucleoprotein complexes containing certain accessory proteins, like WDR43 and NOLC1, enriched for specific subsets of snoRNA-target RNA interactions with distinct roles in ribosome and spliceosome biogenesis. Notably, we discovered that SNORD89 guides 2′-O-methylation at two neighboring sites in U2 snRNA that fine-tune splice site recognition. Conclusions Chimeric eCLIP of snoRNA-associating proteins enables a comprehensive framework for studying snoRNA-target interactions in an RNA-binding protein-dependent manner, revealing novel interactions and regulatory roles in RNA biogenesis.

A small proportion of 4H (Hypomyelination, Hypodontia and Hypogonadotropic Hypogonadism) or RNA polymerase III (POLR3)-related leukodystrophy cases are negative for mutations in the previously identified causative genes POLR3A and POLR3B . Here we report eight of these cases carrying recessive mutations in POLR1C , a gene encoding a shared POLR1 and POLR3 subunit, also mutated in some Treacher Collins syndrome (TCS) cases. Using shotgun proteomics and ChIP sequencing, we demonstrate that leukodystrophy-causative mutations, but not TCS mutations, in POLR1C impair assembly and nuclear import of POLR3, but not POLR1, leading to decreased binding to POLR3 target genes. This study is the first to show that distinct mutations in a gene coding for a shared subunit of two RNA polymerases lead to selective modification of the enzymes’ availability leading to two different clinical conditions and to shed some light on the pathophysiological mechanism of one of the most common hypomyelinating leukodystrophies, POLR3-related leukodystrophy. Some RNA polymerase (POLR) 3-related leukodystrophy cases do not have the causal mutations in POLR3A and POLR3B . Here, by exome sequencing, the authors identify recessive mutations in POLR1C , a gene encoding a shared POLR1 and POLR3 subunit, impairing assembly and nuclear import of POLR3, but not POLR1.

The qkI gene encodes a family of RNA binding proteins alternatively spliced at its 3′ end, giving rise to three major spliced isoforms: QKI-5, QKI-6 and QKI-7. Their expression is tightly regulated during brain development with nuclear QKI-5 being the most abundant during embryogenesis followed by QKI-6 and QKI-7 that peak during myelination. Previously, we generated a mouse conditional qkI allele where exon 2 is excised using Olig2-Cre resulting in QKI-deficient oligodendrocytes (OLs). These mice have dysmyelination and die at the third post-natal week. Herein, we performed a transcriptomic analysis of P14 mouse brains of QKI-proficient ( QKI FL / FL ; - ) and QKI-deficient ( QKI FL / FL ; Olig2 - Cre ) OLs. QKI deficiency results in major global changes of gene expression and RNA processing with >1,800 differentially expressed genes with the top categories being axon ensheathment and myelination. Specific downregulated genes included major myelin proteins, suggesting that the QKI proteins are key regulators of RNA metabolism in OLs. We also identify 810 alternatively spliced genes including known QKI targets, MBP and Nfasc . Interestingly, we observe in QKI FL / FL ; Olig2 - Cre a switch in exon 2-deficient qkI mRNAs favoring the expression of the qkI-5 rather than the qkI-6 and qkI-7 . These findings define QKI as regulators of alternative splicing in OLs including self-splicing.

Hereditary cerebellar ataxias and hereditary spastic paraplegias are clinically and genetically heterogeneous and often overlapping neurological disorders. Mutations in SPG7 cause the autosomal recessive spastic paraplegia type 7 (SPG7), but recent studies indicate that they are also one of the most common causes of recessive cerebellar ataxia. In Quebec, a significant number of patients affected with cerebellar ataxia and spasticity remain without a molecular diagnosis. We performed whole-exome sequencing in three French Canadian (FC) patients affected with spastic ataxia and uncovered compound heterozygous variants in SPG7 in all three. Sanger sequencing of SPG7 exons and exon/intron boundaries was used to screen additional patients. In total, we identified recessive variants in SPG7 in 22 FC patients belonging to 12 families (38.7% of the families screened), including two novel variants. The p.(Ala510Val) variant was the most common in our cohort. Cerebellar features, including ataxia, were more pronounced than spasticity in this cohort. These results strongly suggest that variants affecting the function of SPG7 are the fourth most common form of recessive ataxia in FC patients. Thus, we propose that SPG7 mutations explain a significant proportion of FC spastic ataxia cases and that this gene should be considered in unresolved patients.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS [MIM 270550]) is an early-onset neurodegenerative disorder caused by mutations in the SACS gene. Over 200 SACS mutations have been identified. Most mutations lead to a complete loss of a sacsin, a large 520 kD protein, although some missense mutations are associated with low levels of sacsin expression. We previously showed that Sacs knock-out mice demonstrate early-onset ataxic phenotype with neurofilament bundling in many neuronal populations. To determine if the preservation of some mutated sacsin protein resulted in the same cellular and behavioral alterations, we generated mice expressing an R272C missense mutation, a homozygote mutation found in some affected patients. Though Sacs R272C mice express 21% of wild type brain sacsin and sacsin is found in many neurons, they display similar abnormalities to Sacs knock-out mice, including the development of an ataxic phenotype, reduced Purkinje cell firing rates, and somatodendritic neurofilament bundles in Purkinje cells and other neurons. Together our results support that Sacs missense mutation largely lead to loss of sacsin function.

Recessive mutations in the ubiquitously expressed POLR3A and POLR3B genes are the most common cause of POLR3-related hypomyelinating leukodystrophy (POLR3-HLD), a rare childhood-onset disorder characterized by deficient cerebral myelin formation and cerebellar atrophy. POLR3A and POLR3B encode the two catalytic subunits of RNA Polymerase III (Pol III), which synthesizes numerous small non-coding RNAs. We recently reported that mice homozygous for the Polr3a mutation c.2015G > A (p.Gly672Glu) have no neurological abnormalities and thus do not recapitulate the human POLR3-HLD phenotype. To determine if other POLR3-HLD mutations can cause a leukodystrophy phenotype in mouse, we characterized mice carrying the Polr3b mutation c.308G > A (p.Arg103His). Surprisingly, homozygosity for this mutation was embryonically lethal with only wild-type and heterozygous animals detected at embryonic day 9.5. Using proteomics in a human cell line, we found that the POLR3B R103H mutation severely impairs assembly of the Pol III complex. We next generated Polr3a G672E/G672E /Polr3b +/R103H double mutant mice but observed that this additional mutation was insufficient to elicit a neurological or transcriptional phenotype. Taken together with our previous study on Polr3a G672E mice, our results indicate that missense mutations in Polr3a and Polr3b can variably impair mouse development and Pol III function. Developing a proper model of POLR3-HLD is crucial to gain insights into the pathophysiological mechanisms involved in this devastating neurodegenerative disease.

The ability to conduct image-based, non-invasive cell tagging, independent of genetic engineering, is key to cell biology applications. Here we introduce cell labelling via photobleaching (CLaP), a method that enables instant, specific tagging of individual cells based on a wide array of criteria such as shape, behaviour or positional information. CLaP uses laser illumination to crosslink biotin onto the plasma membrane, coupled with streptavidin conjugates to label individual cells for genomic, cell-tracking, flow cytometry or ultra-microscopy applications. We show that the incorporated mark is stable, non-toxic, retained for several days, and transferred by cell division but not to adjacent cells in culture. To demonstrate the potential of CLaP for genomic applications, we combine CLaP with microfluidics-based single-cell capture followed by transcriptome-wide next-generation sequencing. Finally, we show that CLaP can also be exploited for inducing transient cell adhesion to substrates for microengineering cultures with spatially patterned cell types. Cell labelling in a non-invasive and genetic engineering-free manner is crucial to cell biology applications. Here the authors develop cell labelling via photobleaching (CLaP), that uses laser illumination to label individual cells for genomics, cell-tracking, flow cytometry or ultra-microscopy.

Recessive mutations in the ubiquitously expressed POLR3A gene cause one of the most frequent forms of childhood-onset hypomyelinating leukodystrophy (HLD): POLR3-HLD. POLR3A encodes the largest subunit of RNA Polymerase III (Pol III), which is responsible for the transcription of transfer RNAs (tRNAs) and a large array of other small non-coding RNAs. In order to study the central nervous system pathophysiology of the disease, we introduced the French Canadian founder Polr3a mutation c.2015G > A (p.G672E) in mice, generating homozygous knock-in (KI/KI) as well as compound heterozygous mice for one Polr3a KI and one null allele (KI/KO). Both KI/KI and KI/KO mice are viable and are able to reproduce. To establish if they manifest a motor phenotype, WT, KI/KI and KI/KO mice were submitted to a battery of behavioral tests over one year. The KI/KI and KI/KO mice have overall normal balance, muscle strength and general locomotion. Cerebral and cerebellar Luxol Fast Blue staining and measurement of levels of myelin proteins showed no significant differences between the three groups, suggesting that myelination is not overtly impaired in Polr3a KI/KI and KI/KO mice. Finally, expression levels of several Pol III transcripts in the brain showed no statistically significant differences. We conclude that the first transgenic mice with a leukodystrophy-causing Polr3a mutation do not recapitulate the childhood-onset HLD observed in the majority of human patients with POLR3A mutations, and provide essential information to guide selection of Polr3a mutations for developing future mouse models of the disease.