CoolMPS for robust sequencing of single-nuclear RNAs captured by droplet-based method.

Massively-parallel single-cell and single-nucleus RNA sequencing (scRNA-seq, snRNA-seq) requires extensive sequencing to achieve proper per-cell coverage, making sequencing resources and availability of sequencers critical factors for conducting deep transcriptional profiling. CoolMPS is a novel sequencing-by-synthesis approach that relies on nucleotide labeling by re-usable antibodies, but whether it is applicable to snRNA-seq has not been tested. Here, we use a low-cost and off-the-shelf protocol to chemically convert libraries generated with the widely-used Chromium 10X technology to be sequenceable with CoolMPS technology. To assess the quality and performance of converted libraries sequenced with CoolMPS, we generated a snRNA-seq dataset from the hippocampus of young and old mice. Native libraries were sequenced on an Illumina Novaseq and libraries that were converted to be compatible with CoolMPS were sequenced on a DNBSEQ-400RS. CoolMPS-derived data faithfully replicated key characteristics of the native library dataset, including correct estimation of ambient RNA-contamination, detection of captured cells, cell clustering results, spatial marker gene expression, inter- and intra-replicate differences and gene expression changes during aging. In conclusion, our results show that CoolMPS provides a viable alternative to standard sequencing of RNA from droplet-based libraries.

CIRDES: an efficient genome-wide method for in vivo RNA-RNA interactome analysis.

Complex RNA-RNA interactions underlie fundamental biological processes. However, a large number of RNA-RNA interactions remain unknown. Most existing methods used to map RNA-RNA interactions are based on proximity ligation, but these strategies also capture a huge amount of intramolecular RNA secondary structures, making it almost impossible to detect most RNA-RNA interactions. To overcome this limitation, we developed an efficient, genome-wide method, Capture Interacting RNA and Deep Sequencing (CIRDES) for in vivo capturing of the RNA interactome. We designed multiple 20-nt CIRDES probes tiling the whole RNA sequence of interest. This strategy obtained high selectivity and low background noise proved by qRT-PCR data. CIRDES enriched target RNA and its interacting RNAs from cells crosslinked by formaldehyde in high efficiency. After hybridization and purification, the captured RNAs were converted to the cDNA library after a highly efficient ligation to a 3' end infrared-dye-conjugated RNA adapter based on adapter ligation library construction. Using CIRDES, we detected highly abundant known interacting RNA, as well as a large number of novel targets of U6 snRNA. The enrichment of U4 snRNA, which interacts with U6, confirmed the robustness of the identification of the RNA-RNA interaction by CIRDES. These results suggest that the CIRDES is an efficient strategy for genome-wide RNA-RNA interactome analysis.

High levels of PIWI-interacting RNAs are present in the small RNA landscape of prostate epithelium from vitamin D clinical trial specimens.

BACKGROUND: Vitamin D, a hormone that acts through the nuclear vitamin D receptor (VDR), upregulates antitumorigenic microRNA in prostate epithelium. This may contribute to the lower levels of aggressive prostate cancer (PCa) observed in patients with high serum vitamin D. The small noncoding RNA (ncRNA) landscape includes many other RNA species that remain uncharacterized in prostate epithelium and their potential regulation by vitamin D is unknown. METHODS: Laser capture microdissection (LCM) followed by small-RNA sequencing was used to identify ncRNAs in the prostate epithelium of tissues from a vitamin D-supplementation trial. VDR chromatin immunoprecipitation-sequencing was performed to identify vitamin D genomic targets in primary prostate epithelial cells. RESULTS: Isolation of epithelium by LCM increased sample homogeneity and captured more diversity in ncRNA species compared with publicly available small-RNA sequencing data from benign whole prostate. An abundance of PIWI-interacting RNAs (piRNAs) was detected in normal prostate epithelium. The obligate binding partners of piRNAs, PIWI-like (PIWIL) proteins, were also detected in prostate epithelium. High prostatic vitamin D levels were associated with increased expression of piRNAs. VDR binding sites were located near several ncRNA biogenesis genes and genes regulating translation and differentiation. CONCLUSIONS: Benign prostate epithelium expresses both piRNA and PIWIL proteins, suggesting that these small ncRNA may serve an unknown function in the prostate. Vitamin D may increase the expression of prostatic piRNAs. VDR binding sites in primary prostate epithelial cells are consistent with its reported antitumorigenic functions and a role in ncRNA biogenesis.

sCLIP-an integrated platform to study RNA-protein interactomes in biomedical research: identification of CSTF2tau in alternative processing of small nuclear RNAs.

RNA-binding proteins (RBPs) are central for gene expression by controlling the RNA fate from birth to decay. Various disorders arising from perturbations of RNA-protein interactions document their critical function. However, deciphering their function is complex, limiting the general functional elucidation of this growing class of proteins and their contribution to (patho)physiology. Here, we present sCLIP, a simplified and robust platform for genome-wide interrogation of RNA-protein interactomes based on crosslinking-immunoprecipitation and high-throughput sequencing. sCLIP exploits linear amplification of the immunoprecipitated RNA improving the complexity of the sequencing-library despite significantly reducing the amount of input material and omitting several purification steps. Additionally, it permits a radiolabel-free visualization of immunoprecipitated RNA. In a proof of concept, we identify that CSTF2tau binds many previously not recognized RNAs including histone, snoRNA and snRNAs. CSTF2tau-binding is associated with internal oligoadenylation resulting in shortened snRNA isoforms subjected to rapid degradation. We provide evidence for a new mechanism whereby CSTF2tau controls the abundance of snRNAs resulting in alternative splicing of several RNAs including ANK2 with critical roles in tumorigenesis and cardiac function. Combined with a bioinformatic pipeline sCLIP thus uncovers new functions for established RBPs and fosters the illumination of RBP-protein interaction landscapes in health and disease.

Pseudouridylation meets next-generation sequencing.

The isomerization of uridine to pseudouridine (Ψ), known as pseudouridylation, is the most abundant post-transcriptional modification of stable RNAs. Due to technical limitations in pseudouridine detection methods, studies on pseudouridylation have historically focused on ribosomal RNAs, transfer RNAs, and spliceosomal small nuclear RNAs, where Ψs play a critical role in RNA biogenesis and function. Recently, however, a series of deep sequencing methods-Pseudo-seq, Ψ-seq, PSI-seq, and CeU-seq-has been published to map Ψ positions across the entire transcriptome with single nucleotide resolution. These data have greatly expanded the catalogue of pseudouridylated transcripts, which include messenger RNAs and noncoding RNAs. Furthermore, these methods have revealed conditionally-dependent sites of pseudouridylation that appear in response to cellular stress, suggesting that pseudouridylation may play a role in dynamically modulating RNA function. Collectively, these methods have opened the door to further study of the biological relevance of naturally occurring Ψs. However, an in-depth comparison of these techniques and their results has not yet been undertaken despite all four methods relying on the same basic principle: Ψ detection through selective chemical labeling by the carbodiimide known as CMC. In this article, we will outline the currently available high-throughput Ψ-detection methods and present a comparative analysis of their results. We will then discuss the merits and limitations of these approaches, including those inherent in CMC conjugation, and their potential to further elucidate the function of this ubiquitous and dynamic modification.

Detection and quantification of RNA 2'-O-methylation and pseudouridylation.

RNA-guided RNA modification is a naturally occurring process that introduces 2'-O-methylation and pseudouridylation into rRNA, spliceosomal snRNA and several other types of RNA. The Box C/D ribonucleoproteins (RNP) and Box H/ACA RNP, each containing one unique guide RNA (Box C/D RNA or Box H/ACA RNA) and a set of core proteins, are responsible for 2'-O-methylation and pseudouridylation respectively. Box C/D RNA and Box H/ACA RNA provide the modification specificity through base pairing with their RNA substrate. These post-transcriptional modifications could profoundly alter the properties and functions of substrate RNAs. Thus it is desirable to establish reliable and standardized modification methods to study biological functions of modified nucleotides in RNAs. Here, we present several sensitive and efficient methods and protocols for detecting and quantifying post-transcriptional 2'-O-methylation and pseudouridylation.

Rapid isolation and single-molecule analysis of ribonucleoproteins from cell lysate by SNAP-SiMPull.

Large macromolecular complexes such as the spliceosomal small nuclear ribonucleoproteins (snRNPs) play a variety of roles within the cell. Despite their biological importance, biochemical studies of snRNPs and other machines are often thwarted by practical difficulties in the isolation of sufficient amounts of material. Studies of the snRNPs as well as other macromolecular machines would be greatly facilitated by new approaches that enable their isolation and biochemical characterization. One such approach is single-molecule pull-down (SiMPull) that combines in situ immunopurification of complexes from cell lysates with subsequent single-molecule fluorescence microscopy experiments. We report the development of a new method, called SNAP-SiMPull, that can readily be applied to studies of splicing factors and snRNPs isolated from whole-cell lysates. SNAP-SiMPull overcomes many of the limitations imposed by conventional SiMPull strategies that rely on fluorescent proteins. We have used SNAP-SiMPull to study the yeast branchpoint bridging protein (BBP) as well as the U1 and U6 snRNPs. SNAP-SiMPull will likely find broad use for rapidly isolating complex cellular machines for single-molecule fluorescence colocalization experiments.

Complementation of U4 snRNA in S. cerevisiae splicing extracts for biochemical studies of snRNP assembly and function.

Pre-messenger RNA splicing is a surprisingly complex and dynamic process, the details of which remain largely unknown. One important method for studying splicing involves the replacement of endogenous splicing components with their synthetic counterparts. This enables changes in protein or nucleic acid sequence to be tested for functional effects, as well as the introduction of chemical moieties such as cross-linking groups and fluorescent dyes. To introduce the modified component, the endogenous one must be removed and a method found to reconstitute the active splicing machinery. In extracts prepared from S. cerevisiae, reconstitution has been accomplished with the small, nuclear RNAs U6, U2, and U5.We describe a comparable method to reconstitute active U4 small, nuclear RNA (snRNA) into a splicing extract. In order to remove the endogenous U4 it is necessary to target it for oligo-directed RNase H degradation while active splicing is under way, i.e., in the presence of a splicing transcript and ATP. This allows complete degradation of endogenous U4 and subsequent replacement with an exogenous version. In contrast to the procedures described for depletion of U6, U2, or U5 snRNAs, depletion of U4 requires concurrent active splicing. The ability to reconstitute U4 in yeast extract allows a variety of structural and functional studies to be carried out.

Deep sequencing of small chromatin-associated RNA: isolation and library preparation.

Chromatin-associated RNA (caRNA) is a newly identified class of RNA molecules stably associated with chromatin, maintaining the higher order structure of euchromatic regions in an accessible form (Schubert et al., Mol Cell, doi:10.1016/j.molcel.2012.08.021, 2012). This section provides a detailed protocol describing the isolation of small caRNA from Drosophila cells and preparation of libraries suited for stranded small (20-200 bp) RNA deep sequencing on the Illumina platform.

7SK small nuclear RNA directly affects HMGA1 function in transcription regulation.

Non-coding (nc) RNAs are increasingly recognized to play important regulatory roles in eukaryotic gene expression. The highly abundant and essential 7SK ncRNA has been shown to negatively regulate RNA Polymerase II transcription by inactivating the positive transcription elongation factor b (P-TEFb) in cellular and Tat-dependent HIV transcription. Here, we identify a more general, P-TEFb-independent role of 7SK RNA in directly affecting the function of the architectural transcription factor and chromatin regulator HMGA1. An important regulatory role of 7SK RNA in HMGA1-dependent cell differentiation and proliferation regulation is uncovered with the identification of over 1500 7SK-responsive HMGA1 target genes. Elevated HMGA1 expression is observed in nearly every type of cancer making the use of a 7SK substructure in the inhibition of HMGA1 activity, as pioneered here, potentially useful in therapy. The 7SK-HMGA1 interaction not only adds an essential facet to the comprehension of transcriptional plasticity at the coupling of initiation and elongation, but also might provide a molecular link between HIV reprogramming of cellular gene expression-associated oncogenesis.

In-gel digestion for mass spectrometric characterization of RNA from fluorescently stained polyacrylamide gels.

Although current mass spectrometry-based proteomics technology allows for high-throughput analysis of protein components in functional ribonucleoprotein complexes, this technology has had limited application to studies of RNA itself. Here we present a protocol for RNA analysis using polyacrylamide gel electrophoresis coupled with liquid chromatography-tandem mass spectrometry. Specifically, RNAs of interest are subjected to polyacrylamide gel electrophoresis and stained with a fluorescent dye, and RNAs in gel bands are digested with nuclease and then analyzed directly liquid chromatography-mass spectrometry, resulting in highly accurate mass values and reliable information on post-transcriptional modifications. We demonstrate that the method can be applied to the identification and chemical analysis of small RNAs in mouse embryonic stem cell extracts and of small RNAs in the spliceosomal ribonucleoprotein complex pulled down from yeast cells using a tagged protein cofactor as bait. The protocol is relatively simple and allowed us to identify not only three novel methylated nucleotide residues of RNase P RNA, U6 snRNA, and 7SL RNA prepared from mouse ES cells but also various 3'-end forms of U4, U5S, and U6 snRNAs isolated from the yeast spliceosome at the femtomole level. The method is thus a convenient tool for direct analysis of RNAs in various cellular ribonucleoprotein complexes, particularly for the analysis of post-transcriptional modifications and metabolic processing of RNA.

Quantitative analysis of RNA modifications.

RNA modifications impact numerous cellular processes such as pre-mRNA splicing and protein synthesis. The elucidation of the mechanisms by which these modifications impact cellular processes necessitates the ability to both detect and quantify the presence of these modifications within RNA molecules. Here, we present a detailed procedure that allows the detection and quantification of RNA base modifications. This procedure involves a number of techniques, including oligonucleotide-affinity selection, site-specific cleavage and radiolabeling, nuclease digestion, and thin layer chromatography.

Assembly and glycerol gradient isolation of yeast spliceosomes containing transcribed or synthetic U6 snRNA.

Studies of RNA-protein interactions often require assembly of the RNA-protein complex using in vitro synthesized RNA or recombinant protein. Here, we describe a protocol to assemble a functional spliceosome in yeast extracts using transcribed or synthetic RNAs. The in vitro assembled spliceosome is stable and can be isolated by sedimentation through glycerol gradients for subsequent analysis. The protocols describe two procedures to prepare RNA: using bacteriophage RNA polymerases or ligation of RNA oligos using T4 DNA ligase. We also describe the preparation of splicing competent yeast extracts, the assembly of the spliceosome, and the isolation of the spliceosome by glycerol gradient sedimentation. To allow exogenously added U6 RNA to be incorporated into the spliceosome, the endogenous U6 small nuclear RNA (snRNA) in the extract is eliminated by an antisense U6 DNA oligo and ribonuclease H; a "neutralizing" U6 DNA oligo was then added to protect the incoming U6 RNA. This protocol allows study of the role individual bases or the phosphate backbone of U6 plays in splicing and of the interaction between U6 snRNA and the spliceosomal proteins.

Isolation of an active step I spliceosome and composition of its RNP core.

Formation of catalytically active RNA structures within the spliceosome requires the assistance of proteins. However, little is known about the number and nature of proteins needed to establish and maintain the spliceosome's active site. Here we affinity-purified human spliceosomal C complexes and show that they catalyse exon ligation in the absence of added factors. Comparisons of the composition of the precatalytic versus the catalytic spliceosome revealed a marked exchange of proteins during the transition from the B to the C complex, with apparent stabilization of Prp19-CDC5 complex proteins and destabilization of SF3a/b proteins. Disruption of purified C complexes led to the isolation of a salt-stable ribonucleoprotein (RNP) core that contained both splicing intermediates and U2, U5 and U6 small nuclear RNA plus predominantly U5 and human Prp19-CDC5 proteins and Prp19-related factors. Our data provide insights into the spliceosome's catalytic RNP domain and indicate a central role for the aforementioned proteins in sustaining its catalytically active structure.

Improved identification of enriched peptide RNA cross-links from ribonucleoprotein particles (RNPs) by mass spectrometry.

Direct UV cross-linking combined with mass spectrometry (MS) is a powerful tool to identify hitherto non-characterized protein-RNA contact sites in native ribonucleoprotein particles (RNPs) such as the spliceosome. Identification of contact sites after cross-linking is restricted by: (i) the relatively low cross-linking yield and (ii) the amount of starting material available for cross-linking studies. Therefore, the most critical step in such analyses is the extensive purification of the cross-linked peptide-RNA heteroconjugates from the excess of non-crosslinked material before MS analysis. Here, we describe a strategy that combines small-scale reversed-phase liquid chromatography (RP-HPLC) of UV-irradiated and hydrolyzed RNPs, immobilized metal-ion affinity chromatography (IMAC) to enrich cross-linked species and their analysis by matrix-assisted laser desorption/ionisation (MALDI) MS(/MS). In cases where no MS/MS analysis can be performed, treatment of the enriched fractions with alkaline phosphatase leads to unambiguous identification of the cross-linked species. We demonstrate the feasibility of this strategy by MS analysis of enriched peptide-RNA cross-links from UV-irradiated reconstituted [15.5K-61K-U4atac snRNA] snRNPs and native U1 snRNPs. Applying our approach to a partial complex of U2 snRNP allowed us to identify the contact site between the U2 snRNP-specific protein p14/SF3b14a and the branch-site interacting region (BSiR) of U2 snRNA.

Proteomic analysis of in vivo-assembled pre-mRNA splicing complexes expands the catalog of participating factors.

Previous compositional studies of pre-mRNA processing complexes have been performed in vitro on synthetic pre-mRNAs containing a single intron. To provide a more comprehensive list of polypeptides associated with the pre-mRNA splicing apparatus, we have determined the composition of the bulk pre-mRNA processing machinery in living cells. We purified endogenous nuclear pre-mRNA processing complexes from human and chicken cells comprising the massive (>200S) supraspliceosomes (a.k.a. polyspliceosomes). As expected, RNA components include a heterogeneous mixture of pre-mRNAs and the five spliceosomal snRNAs. In addition to known pre-mRNA splicing factors, 5' end binding factors, 3' end processing factors, mRNA export factors, hnRNPs and other RNA binding proteins, the protein components identified by mass spectrometry include RNA adenosine deaminases and several novel factors. Intriguingly, our purified supraspliceosomes also contain a number of structural proteins, nucleoporins, chromatin remodeling factors and several novel proteins that were absent from splicing complexes assembled in vitro. These in vivo analyses bring the total number of factors associated with pre-mRNA to well over 300, and represent the most comprehensive analysis of the pre-mRNA processing machinery to date.

Identification and analysis of U5 snRNA variants in Drosophila.

Distinct isoforms of spliceosomal RNAs may be involved in regulating pre-messenger RNA splicing in eukaryotic cells. During a large-scale effort to identify small noncoding RNAs in Drosophila, we isolated a U5 snRNA-like molecule containing a 5' segment identical to that of the canonical (major) U5 snRNA but with a variant Sm binding site and a distinct 3' hairpin sequence. Based on this finding, another six similar U5 snRNA-like sequences were identified within the Drosophila genome by sequence similarity to the invariant loop in the 5' half of U5. Interestingly, although all of these variants are expressed in vivo, each shows a distinct temporal expression profile during Drosophila development, and one is expressed primarily in fly heads. The presence of these U5 snRNA variants within RNP particles suggests their role in splicing and implies a possible connection to regulation of developmental and tissue-specific gene expression.

Variants of U1 small nuclear RNA assemble into spliceosomal complexes.

In this study, the existence of additional U1 snRNA variants in the posterior silk gland of the Bombyx mori Nistari strain from India was investigated. Three new U1 variants were detected. One of the new isoforms (U1 SG1) was found to be preferentially assembled into high molecular weight spliceosomal complexes in comparison with the total cellular lysate RNA control. Structural and nucleotide differences were examined in these new isoforms and compared with the previously reported U1 variants. Free energy (Delta G) values for the entire U1 snRNA secondary structures as well as the individual stem/loops (I, II, III and IV) domains of the isoforms were estimated to determine their structural stability.

Purification of functional RNA-protein complexes using MS2-MBP.

Biological machines composed of RNAs and proteins play essential roles in many biological processes. To better understand the mechanism and function of these machines, it is critical to isolate them in a highly purified and functional form. A method for isolating functional RNA-protein complexes assembled in vitro is described. The approach combines gel filtration and an affinity-chromatography strategy using the bacteriophage MS2 coat protein, which binds to a specific RNA-hairpin structure. Using this method, highly purified and functional human spliceosomes have been isolated. The purified spliceosome preparation is used to determine the protein components of the spliceosome by mass spectrometry and to examine the structure of the spliceosome by electron microscopy.