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Background The coronavirus disease 2019 pandemic spread to >200 countries in 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.

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 molecular biology. Here, we present MPRNA-immunoprecipitation (MPRNA-IP), an adaptation of the previously developed massively parallel RNA assay (MPRNA), and a new avenue for in vivo high-throughput dissection of RNA-protein interactions. By using custom pools of tens of thousands of RNA sequences containing systematically designed truncations and mutations, we are 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 NORAD, MS2, and human telomerase RNA, and we describe statistical models for identifying RNA domains and parsing the structural contributions of RNA in these interactions. By blending modern and classical approaches, MPRNA-IP provides a novel high-throughput way to elucidate RNA-based mechanisms behind RNA-protein interactions.

There are currently over 16,000 long-noncoding RNAs (lncRNAs) annotated in mammalian genomes, a number on par with protein-coding genes. Numerous studies have demonstrated that lncRNAs can influence gene expression programs, but lack the temporal resolution to identify the immediate regulatory events. Thus, even for the most well studied the early and primary targets of lncRNAs remains elusive. Here we describe a multifaceted effort to determine the primary targets of a lncRNA and the underlying temporal dynamics. Our approach combines several genetically defined loss-of and gain-of-function, rescue, and inducible lncRNA models. We further resolved gene regulatory events temporally (30 min to four days). Applying this approach, we find that the lncRNA Firre is an RNA-based transcriptional activator. These transcriptional activation events are not mediated by the epigenetic regulators PRC2, WDR5, or G9A, nor are they due to the proximal binding of the Firre lncRNA. Instead, Firre activates transcription by increasing chromatin accessibility (approximately 30 min after induction) and activating primary target genes (about two hours later). Overall, this study suggests Firre functions as an RNA-based transcriptional activator. Citation Format: John L. Rinn, Christian Much, Erika Lasda, Jordan Lewandowski, Michael Smallegan. The lncRNA Firre functions as a transcriptional activator from a distance. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr IA010.

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 1,512 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 2,048 individuals to monitoring the success of on-campus interventions., Graphical abstract Tracking SARS-CoV-2 in on-campus wastewater informs and monitors public health decisions and actions. Image, graphical abstract

Significance We analyzed data from saliva-based COVID-19 screening deployed on the University of Colorado Boulder campus. Our dataset is unique in that all SARS-CoV-2−positive individuals reported no symptoms at the time of saliva collection, and therefore were infected but asymptomatic or presymptomatic. We found that 1) the distribution of viral loads observed in our asymptomatic college population was indistinguishable from what has been reported in hospitalized populations; 2) regardless of symptomatic status, approximately 50% of individuals who test positive for SARS-CoV-2 seem to be in noninfectious phases of the infection; and 3) just 2% of infected individuals carry 90% of the virions circulating within communities, serving as viral “supercarriers” and likely also superspreaders., 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.

In 2019-2020, the COVID-19 pandemic spread to over 200 countries in less than six months. To understand the basis of this aggressive spread, it is essential to determine the transmission rate and define the factors that increase the risk of transmission. One complication is the large fraction of asymptomatic cases, particularly in young populations: these individuals have viral loads indistinguishable from symptomatic people and do transmit the SARS-CoV-2 virus, but they often go undetected. As university students living in residence halls commonly share a small living space with roommates, some schools established regular, high density testing programs to mitigate on-campus spread. In this study, we analyzed longitudinal testing data of residence hall students at the University of Colorado Boulder. We observed that students in single rooms were infected at a lower rate than students in multiple occupancy rooms. However, this was not due to high rates of transmission between roommates, which only occurred approximately 20% of the time. Since these cases were usually asymptomatic at the time of diagnosis, this provides further evidence for asymptomatic transmission. Notably, individuals who likely transmitted to their roommates had an average viral load ∼6.5 times higher than individuals who did not. Although students were moved to separate isolation rooms after diagnosis, there was no difference in time to isolation between these cases with or without transmission. This analysis argues that inter-roommate transmission occurs in a minority of cases in university residence halls and provides strong correlative evidence that viral load can be proportional to the probability of transmission.

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.

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.

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.

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.

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 ∼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.

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 β-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.

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.

The Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII)β has morphogenic functions in neurons not shared by the α isoform. CaMKIIβ contains three exons (v1, v3, and v4) not present in the CaMKIIα gene, and two of these exons (v1 and v4) are subject to differential alternative splicing. We show here that CaMKIIβ, but not α, mediated bundling of F-actin filaments in vitro. Most importantly, inclusion of exon v1 was required for CaMKIIβ association with the F-actin cytoskeleton within cells. CaMKIIβe, which is the dominant variant around birth and lacks exon v1 sequences, failed to associate with F-actin. By contrast, CaMKIIβ′, which instead lacks exon v4, associated with F-actin as full-length CaMKIIβ. Previous studies with CaMKIIβ mutants have indicated a role of nonstimulated kinase activity in enhancing dendritic arborization. Here, we show that F-actin–targeted CaMKIIβ, but not α, was able to phosphorylate actin in vitro even by nonstimulated basal activity in absence of Ca2+/CaM. In rat pancreatic islets and in skeletal muscle, the actin-associated CaMKIIβ′ and βM were the predominant variants, respectively. Thus, cytoskeletal targeting may mediate functions of CaMKIIβ variants also outside the nervous system.

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.

Alternative splicing of mammalian transcripts, which yields many diverse protein products from one gene, is the rule and not the exception. Although the mechanisms that govern alternative splicing are being unraveled, little is known about the evolution of this critical engine of proteome diversity. Here we present a phylogenetic analysis from a sea urchin to humans of the alternative splicing unit encoding the third Ig domain of fibroblast growth factor receptors. The remarkable conservation of intronic control elements, both in structure and function, indicates that the mechanisms that regulate this alternative splicing unit evolved over 600 million years ago.

Protein-RNA interactions play indispensable structural, catalytic, and regulatory roles within the cell. Understanding their physical association in vivo provides valuable insight into their assembly, function, and regulation in the cellular milieu. Inspired by the chromatin immunoprecipitation assay, we have developed a ribonucleoprotein (RNP) immunoprecipitation assay to study RNA-protein interactions in vivo. This method takes advantage of the highly reactive, reversible crosslinker formaldehyde, combined with high-stringency immunoprecipitation to identify specific RNAs associated with a given protein. The RNP immunoprecipitation (RIP) assay was developed using RNA-protein interactions of hepatitis delta virus (HDV) as a model system. HDV is an RNA virus with a single-stranded circular RNA genome that encodes one viral protein, hepatitis delta antigen (HDAg). The high affinity of HDAg for the HDV RNA genome, combined with the well-characterized anti-HDAg antibodies, made this system a logical starting point for the development of the RIP assay. Cells with replicating HDV were crosslinked with formaldehyde and the HDV RNPs were immunoprecipitated using anti-HDAg antibodies. The crosslinks were then reversed by heat treatment, and the immunoprecipitated HDV RNAs were identified by reverse transcription polymerase chain reaction (RT-PCR). The specificity of this assay was tested using HDV mutants and heterologous antibodies for immunoprecipiation followed by RT-PCR with HDV-specific primers. This experiment showed no nonspecific immunoprecipitation of the HDV RNPs. The method was tested further using protein-RNA interactions known to exist in the U1 snRNP. The results indicate that the RIP assay is a powerful tool to identify RNA-protein interactions in vivo and has the potential to unravel the cellular network of RNP complexes in their native setting.

INTRODUCTIONCaenorhabditis elegans is one of a number of organisms in which spliced leader (SL) trans-splicing contributes to the maturation of many pre-mRNA transcripts. C. elegans and related nematodes have two distinct classes of SL RNA—SL1 and SL2—that are used differentially for different classes of pre-mRNAs; namely, SL1 trans-splices non-operon genes and first genes in operons, while SL2 splices downstream operon genes. In vitro RNA splicing has previously been used to analyze substrate RNA sequences, required protein and RNA factors, and splicing mechanisms in a variety of systems. This protocol describes an in vitro splicing assay using C. elegans extract that promotes both cis- and trans-splicing of substrate RNAs. It has the ability to correctly specify and detect SL1 versus SL2 trans-splicing. Operon-derived substrates splice SL2, while substrates with an intron-like sequence followed by a 3′ trans-splice site (outron) splice SL1. Unlabeled T7-transcribed RNA substrates are incubated with crude embryonic extract and trans-spliced by endogenous SL small nuclear ribonucleoproteins (snRNPs). Spliced RNA is analyzed using a reverse transcriptase polymerase chain reaction (RT-PCR). Specific RT and PCR primers ensure detection of trans-spliced substrate RNA and allow a means to distinguish between SL1 and SL2 trans-spliced products. Substrates containing introns are also cis-spliced. All reaction products are ATP-dependent.