Massively parallel dissection of RNA in RNA-protein interactions in vivo.

Many of the biological functions performed by RNA are mediated by RNA-binding proteins (RBPs), and understanding the molecular basis of these interactions is fundamental to biology. Here, we present massively parallel RNA assay combined with immunoprecipitation (MPRNA-IP) for in vivo high-throughput dissection of RNA-protein interactions and describe statistical models for identifying RNA domains and parsing the structural contributions of RNA. By using custom pools of tens of thousands of RNA sequences containing systematically designed truncations and mutations, MPRNA-IP is able to identify RNA domains, sequences, and secondary structures necessary and sufficient for protein binding in a single experiment. We show that this approach is successful for multiple RNAs of interest, including the long noncoding RNA NORAD, bacteriophage MS2 RNA, and human telomerase RNA, and we use it to interrogate the hitherto unknown sequence or structural RNA-binding preferences of the DNA-looping factor CTCF. By integrating systematic mutation analysis with crosslinking immunoprecipitation, MPRNA-IP provides a novel high-throughput way to elucidate RNA-based mechanisms behind RNA-protein interactions in vivo.

Vaccine adjuvant-elicited CD8+ T cell immunity is co-dependent on T-bet and FOXO1.

T-bet and FOXO1 are transcription factors canonically associated with effector and memory T cell fates, respectively. During an infectious response, these factors direct the development of CD8+ T cell fates, where T-bet deficiency leads to ablation of only short-lived effector cells, while FOXO1 deficiency results in selective loss of memory. In contrast, following adjuvanted subunit vaccination in mice, both effector- and memory-fated T cells are compromised in the absence of either T-bet or FOXO1. Thus, unlike responses to challenge with Listeria monocytogenes, productive CD8+ T cell responses to adjuvanted vaccination require coordinated regulation of FOXO1 and T-bet transcriptional programs. Single-cell RNA sequencing analysis confirms simultaneous T-bet, FOXO1, and TCF1 transcriptional activity in vaccine-elicited, but not infection-elicited, T cells undergoing clonal expansion. Collectively, our data show that subunit vaccine adjuvants elicit T cell responses dependent on transcription factors associated with effector and memory cell fates.

High-resolution within-sewer SARS-CoV-2 surveillance facilitates informed intervention.

To assist in the COVID-19 public health guidance on a college campus, daily composite wastewater samples were withdrawn at 20 manhole locations across the University of Colorado Boulder campus. Low-cost autosamplers were fabricated in-house to enable an economical approach to this distributed study. These sample stations operated from August 25th until November 23rd during the fall 2020 semester, with 1512 samples collected. The concentration of SARS-CoV-2 in each sample was quantified through two comparative reverse transcription quantitative polymerase chain reactions (RT-qPCRs). These methods were distinct in the utilization of technical replicates and normalization to an endogenous control. (1) Higher temporal resolution compensates for supply chain or other constraints that prevent technical or biological replicates. (2) The data normalized by an endogenous control agreed with the raw concentration data, minimizing the utility of normalization. The raw wastewater concentration values reflected SARS-CoV-2 prevalence on campus as detected by clinical services. Overall, combining the low-cost composite sampler with a method that quantifies the SARS-CoV-2 signal within six hours enabled actionable and time-responsive data delivered to key stakeholders. With daily reporting of the findings, wastewater surveillance assisted in decision making during critical phases of the pandemic on campus, from detecting individual cases within populations ranging from 109 to 2048 individuals to monitoring the success of on-campus interventions.

Higher Viral Load Drives Infrequent Severe Acute Respiratory Syndrome Coronavirus 2 Transmission Between Asymptomatic Residence Hall Roommates.

BACKGROUND: The coronavirus disease 2019 pandemic spread to >200 countries in <6 months. To understand coronavirus spread, determining transmission rate and defining factors that increase transmission risk are essential. Most cases are asymptomatic, but people with asymptomatic infection have viral loads indistinguishable from those in symptomatic people, and they do transmit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, asymptomatic cases are often undetected. METHODS: Given high residence hall student density, the University of Colorado Boulder established a mandatory weekly screening test program. We analyzed longitudinal data from 6408 students and identified 116 likely transmission events in which a second roommate tested positive within 14 days of the index roommate. RESULTS: Although the infection rate was lower in single-occupancy rooms (10%) than in multiple-occupancy rooms (19%), interroommate transmission occurred only about 20% of the time. Cases were usually asymptomatic at the time of detection. Notably, individuals who likely transmitted had an average viral load approximately 6.5-fold higher than individuals who did not (mean quantification cycle [Cq], 26.2 vs 28.9). Although students with diagnosed SARS-CoV-2 infection moved to isolation rooms, there was no difference in time to isolation between cases with or without interroommate transmission. CONCLUSIONS: This analysis argues that interroommate transmission occurs infrequently in residence halls and provides strong correlative evidence that viral load is proportional to transmission probability.

Just 2% of SARS-CoV-2-positive individuals carry 90% of the virus circulating in communities.

We analyze data from the fall 2020 pandemic response efforts at the University of Colorado Boulder, where more than 72,500 saliva samples were tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using qRT-PCR. All samples were collected from individuals who reported no symptoms associated with COVID-19 on the day of collection. From these, 1,405 positive cases were identified. The distribution of viral loads within these asymptomatic individuals was indistinguishable from what has been previously observed in symptomatic individuals. Regardless of symptomatic status, ∼50% of individuals who test positive for SARS-CoV-2 seem to be in noninfectious phases of the disease, based on having low viral loads in a range from which live virus has rarely been isolated. We find that, at any given time, just 2% of individuals carry 90% of the virions circulating within communities, serving as viral "supercarriers" and possibly also superspreaders.

Saliva TwoStep for rapid detection of asymptomatic SARS-CoV-2 carriers.

Here, we develop a simple molecular test for SARS-CoV-2 in saliva based on reverse transcription loop-mediated isothermal amplification. The test has two steps: (1) heat saliva with a stabilization solution and (2) detect virus by incubating with a primer/enzyme mix. After incubation, saliva samples containing the SARS-CoV-2 genome turn bright yellow. Because this test is pH dependent, it can react falsely to some naturally acidic saliva samples. We report unique saliva stabilization protocols that rendered 295 healthy saliva samples compatible with the test, producing zero false positives. We also evaluated the test on 278 saliva samples from individuals who were infected with SARS-CoV-2 but had no symptoms at the time of saliva collection, and from 54 matched pairs of saliva and anterior nasal samples from infected individuals. The Saliva TwoStep test described herein identified infections with 94% sensitivity and >99% specificity in individuals with sub-clinical (asymptomatic or pre-symptomatic) infections.

Just 2% of SARS-CoV-2-positive individuals carry 90% of the virus circulating in communities.

We analyze data from the Fall 2020 pandemic response efforts at the University of Colorado Boulder (USA), where more than 72,500 saliva samples were tested for SARS-CoV-2 using quantitative RT-PCR. All samples were collected from individuals who reported no symptoms associated with COVID-19 on the day of collection. From these, 1,405 positive cases were identified. The distribution of viral loads within these asymptomatic individuals was indistinguishable from what has been previously reported in symptomatic individuals. Regardless of symptomatic status, approximately 50% of individuals who test positive for SARS-CoV-2 seem to be in non-infectious phases of the disease, based on having low viral loads in a range from which live virus has rarely been isolated. We find that, at any given time, just 2% of individuals carry 90% of the virions circulating within communities, serving as viral "super-carriers" and possibly also super-spreaders.

Saliva TwoStep for rapid detection of asymptomatic SARS-CoV-2 carriers.

Here, we develop a simple molecular test for SARS-CoV-2 in saliva based on reverse transcription loop-mediated isothermal amplification (RT-LAMP). The test has two steps: 1) heat saliva with a stabilization solution, and 2) detect virus by incubating with a primer/enzyme mix. After incubation, saliva samples containing the SARS-CoV-2 genome turn bright yellow. Because this test is pH dependent, it can react falsely to some naturally acidic saliva samples. We report unique saliva stabilization protocols that rendered 295 healthy saliva samples compatible with the test, producing zero false positives. We also evaluated the test on 278 saliva samples from individuals who were infected with SARS-CoV-2 but had no symptoms at the time of saliva collection, and from 54 matched pairs of saliva and anterior nasal samples from infected individuals. The Saliva TwoStep test described herein identified infections with 94% sensitivity and >99% specificity in individuals with sub-clinical (asymptomatic or pre-symptomatic) infections.

Analysis of the CaMKIIα and ß splice-variant distribution among brain regions reveals isoform-specific differences in holoenzyme formation.

Four CaMKII isoforms are encoded by distinct genes, and alternative splicing within the variable linker-region generates additional diversity. The α and ß isoforms are largely brain-specific, where they mediate synaptic functions underlying learning, memory and cognition. Here, we determined the α and ß splice-variant distribution among different mouse brain regions. Surprisingly, the nuclear variant αB was detected in all regions, and even dominated in hypothalamus and brain stem. For CaMKIIß, the full-length variant dominated in most regions (with higher amounts of minor variants again seen in hypothalamus and brain stem). The mammalian but not fish CaMKIIß gene lacks exon v3N that encodes the nuclear localization signal in αB, but contains three exons not found in the CaMKIIα gene (exons v1, v4, v5). While skipping of exons v1 and/or v5 generated the minor splice-variants ß', ße and ße', essentially all transcripts contained exon v4. However, we instead detected another minor splice-variant (now termed ßH), which lacks part of the hub domain that mediates formation of CaMKII holoenzymes. Surprisingly, in an optogenetic cellular assay of protein interactions, CaMKIIßH was impaired for binding to the ß hub domain, but still bound CaMKIIα. This provides the first indication for isoform-specific differences in holoenzyme formation.

Circular RNAs Co-Precipitate with Extracellular Vesicles: A Possible Mechanism for circRNA Clearance.

Backspliced circular RNAs (circRNAs) are prevalent in many eukaryotic systems and are spliced from thousands of different genes. Where examined, circRNAs are often highly stable and the mechanisms by which cells degrade and/or clear circRNAs from the cells are unknown. Here we investigated the possibility that cells can eliminate circRNAs into extracellular space, possibly within released vesicles such as exosomes and microvesicles. From three different cell lines and examining multiple circRNAs, we show that extracellular vesicle (EVs) preparations recovered from cell culture conditioned media contain established circRNAs. Moreover, these circRNAs are enriched over their linear counterparts within EV preparations when compared to the producing cells. This supports the idea that expulsion from cells into extracellular space, as by EVs release, can be a mechanism by which cells clear circRNAs. Moreover, since EVs can be taken up by other cells, excreted circRNAs could contribute to cell to cell communication.

Circular RNAs: diversity of form and function.

It is now clear that there is a diversity of circular RNAs in biological systems. Circular RNAs can be produced by the direct ligation of 5' and 3' ends of linear RNAs, as intermediates in RNA processing reactions, or by "backsplicing," wherein a downstream 5' splice site (splice donor) is joined to an upstream 3' splice site (splice acceptor). Circular RNAs have unique properties including the potential for rolling circle amplification of RNA, the ability to rearrange the order of genomic information, protection from exonucleases, and constraints on RNA folding. Circular RNAs can function as templates for viroid and viral replication, as intermediates in RNA processing reactions, as regulators of transcription in cis, as snoRNAs, and as miRNA sponges. Herein, we review the breadth of circular RNAs, their biogenesis and metabolism, and their known and anticipated functions.

Trans-splicing.

Trans-splicing is the joining together of portions of two separate pre-mRNA molecules. The two distinct categories of spliceosomal trans-splicing are genic trans-splicing, which joins exons of different pre-mRNA transcripts, and spliced leader (SL) trans-splicing, which involves an exon donated from a specialized SL RNA. Both depend primarily on the same signals and components as cis-splicing. Genic trans-splicing events producing protein-coding mRNAs have been described in a variety of organisms, including Caenorhabditis elegans and Drosophila. In mammalian cells, genic trans-splicing can be associated with cancers and translocations. SL trans-splicing has mainly been studied in nematodes and trypanosomes, but there are now numerous and diverse phyla (including primitive chordates) where this type of trans-splicing has been detected. Such diversity raises questions as to the evolutionary origin of the process. Another intriguing question concerns the function of trans-splicing, as operon resolution can only account for a small proportion of the total amount of SL trans-splicing.

Polycistronic pre-mRNA processing in vitro: snRNP and pre-mRNA role reversal in trans-splicing.

Spliced leader (SL) trans-splicing in Caenorhabditis elegans attaches a 22-nucleotide (nt) exon onto the 5' end of many mRNAs. A particular class of SL, SL2, splices mRNAs of downstream operon genes. Here we use an embryonic extract-based in vitro splicing system to show that SL2 specificity information is encoded within the polycistronic pre-mRNA, and that trans-splicing specificity is recapitulated in vitro. We define an RNA sequence required for SL2 trans-splicing, the U-rich (Ur) element, through mutational analysis and bioinformatics as a short stem-loop followed by a sequence motif, UAYYUU, located approximately 50 nt upstream of the trans-splice site. Furthermore, this element is predicted in intercistronic regions of numerous operons of C. elegans and other species that use SL2 trans-splicing. We propose that the UAYYUU motif hybridizes with the 5' splice site on the SL2 RNA to recruit the SL to the pre-mRNA. In this way, the UAYYUU motif in the pre-mRNA would serve an analogous function to the similar sequence in the U1 snRNA, which binds to the 5' splice site of introns, effectively reversing the roles of snRNP and pre-mRNA in trans-splicing.

Identification of an intronic splicing enhancer essential for the inclusion of FGFR2 exon IIIc.

The ligand specificity of fibroblast growth factor receptor 2 (FGFR2) is determined by the alternative splicing of exons 8 (IIIb) or 9 (IIIc). Exon IIIb is included in epithelial cells, whereas exon IIIc is included in mesenchymal cells. Although a number of cis elements and trans factors have been identified that play a role in exon IIIb inclusion in epithelium, little is known about the activation of exon IIIc in mesenchyme. We report here the identification of a splicing enhancer required for IIIc inclusion. This 24-nucleotide (nt) downstream intronic splicing enhancer (DISE) is located within intron 9 immediately downstream of exon IIIc. DISE was able to activate the inclusion of heterologous exons rat FGFR2 IIIb and human beta-globin exon 2 in cell lines from different tissues and species and also in HeLa cell nuclear extracts in vitro. DISE was capable of replacing the intronic activator sequence 1 (IAS1), a known IIIb splicing enhancer and vice versa. This fact, together with the requirement for DISE to be close to the 5'-splice site and the ability of DISE to promote binding of U1 snRNP, suggested that IAS1 and DISE belong to the same class of cis-acting elements.

A novel family of C. elegans snRNPs contains proteins associated with trans-splicing.

In many Caenorhabditis elegans pre-mRNAs, the RNA sequence between the 5' cap and the first 3' splice site is replaced by trans-splicing a short spliced leader (SL) from the Sm snRNP, SL1. C. elegans also utilizes a similar Sm snRNP, SL2, to trans-splice at sites between genes in polycistronic pre-mRNAs from operons. How do SL1 and SL2 snRNPs function in different contexts? Here we show that the SL1 snRNP contains a complex of SL75p and SL21p, which are homologs of novel proteins previously reported in the Ascaris SL snRNP. Interestingly, we show that the SL2 snRNP does not contain these proteins. However, SL75p and SL26p, a paralog of SL21p, are components of another Sm snRNP that contains a novel snRNA species, Sm Y. Knockdown of SL75p is lethal. However, knockdown of either SL21p or SL26p alone leads to cold-sensitive sterility, whereas knockdown of both SL21p and SL26p is lethal. This suggests that these two proteins have overlapping functions even though they are associated with different classes of snRNP. These phenotypic relationships, along with the association of SL26p with SL75p, imply that, like the SL1 RNA/Sm/SL75p/SL21p complex, the Sm Y/Sm/SL75p/SL26p complex is associated with trans-splicing.

CaMKIIbeta association with the actin cytoskeleton is regulated by alternative splicing.

The Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII)beta has morphogenic functions in neurons not shared by the alpha isoform. CaMKIIbeta contains three exons (v1, v3, and v4) not present in the CaMKIIalpha gene, and two of these exons (v1 and v4) are subject to differential alternative splicing. We show here that CaMKIIbeta, but not alpha, mediated bundling of F-actin filaments in vitro. Most importantly, inclusion of exon v1 was required for CaMKIIbeta association with the F-actin cytoskeleton within cells. CaMKIIbetae, which is the dominant variant around birth and lacks exon v1 sequences, failed to associate with F-actin. By contrast, CaMKIIbeta', which instead lacks exon v4, associated with F-actin as full-length CaMKIIbeta. Previous studies with CaMKIIbeta mutants have indicated a role of nonstimulated kinase activity in enhancing dendritic arborization. Here, we show that F-actin-targeted CaMKIIbeta, but not alpha, was able to phosphorylate actin in vitro even by nonstimulated basal activity in absence of Ca(2+)/CaM. In rat pancreatic islets and in skeletal muscle, the actin-associated CaMKIIbeta' and betaM were the predominant variants, respectively. Thus, cytoskeletal targeting may mediate functions of CaMKIIbeta variants also outside the nervous system.

Embryonic and larval expression of zebrafish voltage-gated sodium channel alpha-subunit genes.

Whereas it is known that voltage-gated calcium channels play important roles during development, potential embryonic roles of voltage-gated sodium channels have received much less attention. Voltage-gated sodium channels consist of pore-forming alpha-subunits (Na(v)1) and auxiliary beta-subunits. Here, we report the embryonic and larval expression patterns for all eight members of the gene family (scna) coding for zebrafish Na(v)1 proteins. We find that each scna gene displays a distinct expression pattern that is temporally and spatially dynamic during embryonic and larval stages. Overall, our findings indicate that scna gene expression occurs sufficiently early during embryogenesis to play developmental roles for both muscle and nervous tissues.

Alternative splicing in disease and therapy.

Alternative splicing is the major source of proteome diversity in humans and thus is highly relevant to disease and therapy. For example, recent work suggests that the long-sought-after target of the analgesic acetaminophen is a neural-specific, alternatively spliced isoform of cyclooxygenase 1 (COX-1). Several important diseases, such as cystic fibrosis, have been linked with mutations or variations in either cis-acting elements or trans-acting factors that lead to aberrant splicing and abnormal protein production. Correction of erroneous splicing is thus an important goal of molecular therapies. Recent experiments have used modified oligonucleotides to inhibit cryptic exons or to activate exons weakened by mutations, suggesting that these reagents could eventually lead to effective therapies.

A stem structure in fibroblast growth factor receptor 2 transcripts mediates cell-type-specific splicing by approximating intronic control elements.

Alternative splicing of fibroblast growth factor receptor 2 (FGFR2) occurs in a cell-type-specific manner with the mutually exclusive use of exon IIIb or exon IIIc. Specific inclusion of exon IIIb is observed in epithelial cells, whereas exon IIIc inclusion is seen in mesenchymal cells. Epithelium-specific activation of exon IIIb and repression of exon IIIc are coordinately regulated by intronic activating sequence 2 (IAS2) and intronic splicing activator and repressor (ISAR) elements in FGFR2 pre-mRNA. Previously, it has been suggested that IAS2 and a 20-nucleotide core sequence of ISAR form a stem structure that allows for the proper regulation of FGFR2 alternative splicing. Replacement of IAS2 and the ISAR core with random sequences capable of stem formation resulted in the proper activation of exon IIIb and repression of exon IIIc in epithelial cells. Given the high degree of phylogenetic conservation of the IAS2-ISAR core structure and the fact that unrelated stem-forming sequences could functionally substitute for IAS2 and ISAR elements, we postulated that the stem structure facilitated the approximation of intronic control elements. Indeed, deletion of the entire stem-loop region and juxtaposition of sequences immediately upstream of IAS2 with sequences immediately downstream of the ISAR core maintained proper cell-type-specific inclusion of exon IIIb. These data demonstrate that IAS2 and the ISAR core are dispensable for the cell-type-specific activation of exon IIIb; thus, the major, if not the sole, role of the IAS2-ISAR stem in exon IIIb activation is to approximate sequences upstream of IAS2 with sequences downstream of the ISAR core. The downstream sequence is very likely a highly conserved GCAUG element, which we show was required for efficient exon IIIb activation.