Expanded palette of RNA base editors for comprehensive RBP-RNA interactome studies.

RNA binding proteins (RBPs) are key regulators of RNA processing and cellular function. Technologies to discover RNA targets of RBPs such as TRIBE (targets of RNA binding proteins identified by editing) and STAMP (surveying targets by APOBEC1 mediated profiling) utilize fusions of RNA base-editors (rBEs) to RBPs to circumvent the limitations of immunoprecipitation (CLIP)-based methods that require enzymatic digestion and large amounts of input material. To broaden the repertoire of rBEs suitable for editing-based RBP-RNA interaction studies, we have devised experimental and computational assays in a framework called PRINTER (protein-RNA interaction-based triaging of enzymes that edit RNA) to assess over thirty A-to-I and C-to-U rBEs, allowing us to identify rBEs that expand the characterization of binding patterns for both sequence-specific and broad-binding RBPs. We also propose specific rBEs suitable for dual-RBP applications. We show that the choice between single or multiple rBEs to fuse with a given RBP or pair of RBPs hinges on the editing biases of the rBEs and the binding preferences of the RBPs themselves. We believe our study streamlines and enhances the selection of rBEs for the next generation of RBP-RNA target discovery.

circHIPK3 nucleates IGF2BP2 and functions as a competing endogenous RNA.

Circular RNAs (circRNAs) represent a class of widespread endogenous RNAs that regulate gene expression and thereby influence cell biological decisions with implications for the pathogenesis of several diseases. Here, we disclose a novel gene-regulatory role of circHIPK3 by combining analyses of large genomics datasets and mechanistic cell biological follow-up experiments. Specifically, we use temporal depletion of circHIPK3 or specific RNA binding proteins (RBPs) and identify several perturbed genes by RNA sequencing analyses. Using expression-coupled motif analyses of mRNA expression data from various knockdown experiments, we identify an 11-mer motif within circHIPK3, which is also enriched in genes that become downregulated upon circHIPK3 depletion. By mining eCLIP datasets, we find that the 11-mer motif constitutes a strong binding site for IGF2BP2 and validate this circHIPK3-IGF2BP2 interaction experimentally using RNA-immunoprecipitation and competition assays in bladder cancer cell lines. Our results suggest that circHIPK3 and IGF2BP2 mRNA targets compete for binding. Since the identified 11-mer motif found in circHIPK3 is enriched in upregulated genes following IGF2BP2 knockdown, and since IGF2BP2 depletion conversely globally antagonizes the effect of circHIPK3 knockdown on target genes, our results suggest that circHIPK3 can sequester IGF2BP2 as a competing endogenous RNA (ceRNA), leading to target mRNA stabilization. As an example of a circHIPK3-regulated gene, we focus on the STAT3 mRNA as a specific substrate of IGF2BP2 and validate that manipulation of circHIPK3 regulates IGF2BP2-STAT3 mRNA binding and thereby STAT3 mRNA levels. However, absolute copy number quantifications demonstrate that IGF2BP2 outnumbers circHIPK3 by orders of magnitude, which is inconsistent with a simple 1:1 ceRNA hypothesis. Instead, we show that circHIPK3 can nucleate multiple copies of IGF2BP2, potentially via phase separation, to produce IGF2BP2 condensates. Finally, we show that circHIPK3 expression correlates with overall survival of patients with bladder cancer. Our results are consistent with a model where relatively few cellular circHIPK3 molecules function as inducers of IGF2BP2 condensation thereby regulating STAT3 and other key factors for cell proliferation and potentially cancer progression.

Expanded palette of RNA base editors for comprehensive RBP-RNA interactome studies.

RNA binding proteins (RBPs) are key regulators of RNA processing and cellular function. Technologies to discover RNA targets of RBPs such as TRIBE (targets of RNA binding proteins identified by editing) and STAMP (surveying targets by APOBEC1 mediated profiling) utilize fusions of RNA base-editors (rBEs) to RBPs to circumvent the limitations of immunoprecipitation (CLIP)-based methods that require enzymatic digestion and large amounts of input material. To broaden the repertoire of rBEs suitable for editing-based RBP-RNA interaction studies, we have devised experimental and computational assays in a framework called PRINTER (protein-RNA interaction-based triaging of enzymes that edit RNA) to assess over thirty A-to-I and C-to-U rBEs, allowing us to identify rBEs that expand the characterization of binding patterns for both sequence-specific and broad-binding RBPs. We also propose specific rBEs suitable for dual-RBP applications. We show that the choice between single or multiple rBEs to fuse with a given RBP or pair of RBPs hinges on the editing biases of the rBEs and the binding preferences of the RBPs themselves. We believe our study streamlines and enhances the selection of rBEs for the next generation of RBP-RNA target discovery.

HydRA: Deep-learning models for predicting RNA-binding capacity from protein interaction association context and protein sequence.

RNA-binding proteins (RBPs) control RNA metabolism to orchestrate gene expression and, when dysfunctional, underlie human diseases. Proteome-wide discovery efforts predict thousands of RBP candidates, many of which lack canonical RNA-binding domains (RBDs). Here, we present a hybrid ensemble RBP classifier (HydRA), which leverages information from both intermolecular protein interactions and internal protein sequence patterns to predict RNA-binding capacity with unparalleled specificity and sensitivity using support vector machines (SVMs), convolutional neural networks (CNNs), and Transformer-based protein language models. Occlusion mapping by HydRA robustly detects known RBDs and predicts hundreds of uncharacterized RNA-binding associated domains. Enhanced CLIP (eCLIP) for HydRA-predicted RBP candidates reveals transcriptome-wide RNA targets and confirms RNA-binding activity for HydRA-predicted RNA-binding associated domains. HydRA accelerates construction of a comprehensive RBP catalog and expands the diversity of RNA-binding associated domains.

Discovery and functional interrogation of SARS-CoV-2 protein-RNA interactions.

The COVID-19 pandemic is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The betacoronvirus has a positive sense RNA genome which encodes for several RNA binding proteins. Here, we use enhanced crosslinking and immunoprecipitation to investigate SARS-CoV-2 protein interactions with viral and host RNAs in authentic virus-infected cells. SARS-CoV-2 proteins, NSP8, NSP12, and nucleocapsid display distinct preferences to specific regions in the RNA viral genome, providing evidence for their shared and separate roles in replication, transcription, and viral packaging. SARS-CoV-2 proteins expressed in human lung epithelial cells bind to 4773 unique host coding RNAs. Nine SARS-CoV-2 proteins upregulate target gene expression, including NSP12 and ORF9c, whose RNA substrates are associated with pathways in protein N-linked glycosylation ER processing and mitochondrial processes. Furthermore, siRNA knockdown of host genes targeted by viral proteins in human lung organoid cells identify potential antiviral host targets across different SARS-CoV-2 variants. Conversely, NSP9 inhibits host gene expression by blocking mRNA export and dampens cytokine productions, including interleukin-1α/ß. Our viral protein-RNA interactome provides a catalog of potential therapeutic targets and offers insight into the etiology of COVID-19 as a safeguard against future pandemics.

Transcriptome-wide identification of RNA-binding protein binding sites using seCLIP-seq.

Discovery of interaction sites between RNA-binding proteins (RBPs) and their RNA targets plays a critical role in enabling our understanding of how these RBPs control RNA processing and regulation. Cross-linking and immunoprecipitation (CLIP) provides a generalizable, transcriptome-wide method by which RBP/RNA complexes are purified and sequenced to identify sites of intermolecular contact. By simplifying technical challenges in prior CLIP methods and incorporating the generation of and quantitative comparison against size-matched input controls, the single-end enhanced CLIP (seCLIP) protocol allows for the profiling of these interactions with high resolution, efficiency and scalability. Here, we present a step-by-step guide to the seCLIP method, detailing critical steps and offering insights regarding troubleshooting and expected results while carrying out the ~4-d protocol. Furthermore, we describe a comprehensive bioinformatics pipeline that offers users the tools necessary to process two replicate datasets and identify reproducible and significant peaks for an RBP of interest in ~2 d.

Discovery and functional interrogation of SARS-CoV-2 protein-RNA interactions.

The COVID-19 pandemic is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The betacoronvirus has a positive sense RNA genome which encodes for several RNA binding proteins. Here, we use enhanced crosslinking and immunoprecipitation to investigate SARS-CoV-2 protein interactions with viral and host RNAs in authentic virus-infected cells. SARS-CoV-2 proteins, NSP8, NSP12, and nucleocapsid display distinct preferences to specific regions in the RNA viral genome, providing evidence for their shared and separate roles in replication, transcription, and viral packaging. SARS-CoV-2 proteins expressed in human lung epithelial cells bind to 4773 unique host coding RNAs. Nine SARS-CoV-2 proteins upregulate target gene expression, including NSP12 and ORF9c, whose RNA substrates are associated with pathways in protein N-linked glycosylation ER processing and mitochondrial processes. Furthermore, siRNA knockdown of host genes targeted by viral proteins in human lung organoid cells identify potential antiviral host targets across different SARS-CoV-2 variants. Conversely, NSP9 inhibits host gene expression by blocking mRNA export and dampens cytokine productions, including interleukin-1α/ß. Our viral protein-RNA interactome provides a catalog of potential therapeutic targets and offers insight into the etiology of COVID-19 as a safeguard against future pandemics.

Integrative RNA-omics Discovers GNAS Alternative Splicing as a Phenotypic Driver of Splicing Factor-Mutant Neoplasms.

Mutations in splicing factors (SF) are the predominant class of mutations in myelodysplastic syndrome (MDS), but convergent downstream disease drivers remain elusive. To identify common direct targets of missplicing by mutant U2AF1 and SRSF2, we performed RNA sequencing and enhanced version of the cross-linking and immunoprecipitation assay in human hematopoietic stem/progenitor cells derived from isogenic induced pluripotent stem cell (iPSC) models. Integrative analyses of alternative splicing and differential binding converged on a long isoform of GNAS (GNAS-L), promoted by both mutant factors. MDS population genetics, functional and biochemical analyses support that GNAS-L is a driver of MDS and encodes a hyperactive long form of the stimulatory G protein alpha subunit, Gαs-L, that activates ERK/MAPK signaling. SF-mutant MDS cells have activated ERK signaling and consequently are sensitive to MEK inhibitors. Our findings highlight an unexpected and unifying mechanism by which SRSF2 and U2AF1 mutations drive oncogenesis with potential therapeutic implications for MDS and other SF-mutant neoplasms. SIGNIFICANCE: SF mutations are disease-defining in MDS, but their critical effectors remain unknown. We discover the first direct target of convergent missplicing by mutant U2AF1 and SRSF2, a long GNAS isoform, which activates G protein and ERK/MAPK signaling, thereby driving MDS and rendering mutant cells sensitive to MEK inhibition. This article is highlighted in the In This Issue feature, p. 587.

Transcriptome-wide profiles of circular RNA and RNA-binding protein interactions reveal effects on circular RNA biogenesis and cancer pathway expression.

BACKGROUND: Circular RNAs (circRNAs) are stable, often highly expressed RNA transcripts with potential to modulate other regulatory RNAs. A few circRNAs have been shown to bind RNA-binding proteins (RBPs); however, little is known about the prevalence and distribution of these interactions in different biological contexts. METHODS: We conduct an extensive screen of circRNA-RBP interactions in the ENCODE cell lines HepG2 and K562. We profile circRNAs in deep-sequenced total RNA samples and analyze circRNA-RBP interactions using a large set of eCLIP data with binding sites of 150 RBPs. We validate interactions for select circRNAs and RBPs by performing RNA immunoprecipitation and functionally characterize our most interesting candidates by conducting knockdown studies followed by RNA-Seq. RESULTS: We generate a comprehensive catalog of circRNA-RBP interactions in HepG2 and K562 cells. We show that KHSRP binding sites are enriched in flanking introns of circRNAs and that KHSRP depletion affects circRNA biogenesis. We identify circRNAs that are highly covered by RBP binding sites and experimentally validate individual circRNA-RBP interactions. We show that circCDYL, a highly expressed circRNA with clinical and functional implications in bladder cancer, is almost completely covered with GRWD1 binding sites in HepG2 cells, and that circCDYL depletion counteracts the effect of GRWD1 depletion. Furthermore, we confirm interactions between circCDYL and RBPs in bladder cancer cells and demonstrate that circCDYL depletion affects hallmarks of cancer and perturbs the expression of key cancer genes, e.g., TP53. Finally, we show that elevated levels of circCDYL are associated with overall survival of bladder cancer patients. CONCLUSIONS: Our study demonstrates transcriptome-wide and cell-type-specific circRNA-RBP interactions that could play important regulatory roles in tumorigenesis.

Principles of RNA processing from analysis of enhanced CLIP maps for 150 RNA binding proteins.

BACKGROUND: A critical step in uncovering rules of RNA processing is to study the in vivo regulatory networks of RNA binding proteins (RBPs). Crosslinking and immunoprecipitation (CLIP) methods enable mapping RBP targets transcriptome-wide, but methodological differences present challenges to large-scale analysis across datasets. The development of enhanced CLIP (eCLIP) enabled the mapping of targets for 150 RBPs in K562 and HepG2, creating a unique resource of RBP interactomes profiled with a standardized methodology in the same cell types. RESULTS: Our analysis of 223 eCLIP datasets reveals a range of binding modalities, including highly resolved positioning around splicing signals and mRNA untranslated regions that associate with distinct RBP functions. Quantification of enrichment for repetitive and abundant multicopy elements reveals 70% of RBPs have enrichment for non-mRNA element classes, enables identification of novel ribosomal RNA processing factors and sites, and suggests that association with retrotransposable elements reflects multiple RBP mechanisms of action. Analysis of spliceosomal RBPs indicates that eCLIP resolves AQR association after intronic lariat formation, enabling identification of branch points with single-nucleotide resolution, and provides genome-wide validation for a branch point-based scanning model for 3' splice site recognition. Finally, we show that eCLIP peak co-occurrences across RBPs enable the discovery of novel co-interacting RBPs. CONCLUSIONS: This work reveals novel insights into RNA biology by integrated analysis of eCLIP profiling of 150 RBPs with distinct functions. Further, our quantification of both mRNA and other element association will enable further research to identify novel roles of RBPs in regulating RNA processing.

Counseling services for Asian, Latino/a, and White American students: Initial severity, session attendance, and outcome.

OBJECTIVE: The current study examined racial/ethnic differences in initial severity, session attendance, and counseling outcomes in a large and diverse sample of Asian American, Latino/a, and White student clients who utilized university counseling services between 2008 and 2012. METHOD: We used archival data of 5,472 clients (62% female; M age = 23.1, SD = 4.3) who self-identified their race/ethnicity as being Asian American (38.9%), Latino/a (14.9%), or White (46.2%). Treatment engagement was measured by the number of counseling sessions attended; initial severity and treatment outcome were measured using the Outcome Questionnaire-45. RESULTS: Asian American clients, particularly Chinese, Filipino/a, Korean, and Vietnamese Americans, had greater initial severity compared with White clients. Asian Indian, Korean, and Vietnamese American clients used significantly fewer sessions of counseling than White clients after controlling for initial severity. All racial/ethnic minority groups continued to have clinically significant distress in certain areas (e.g., social role functioning) at counseling termination. CONCLUSIONS: These findings highlight the need to devote greater attention to the counseling experiences of racial/ethnic minority clients, especially certain Asian American groups. Further research directions are provided. (PsycINFO Database Record

Increasingly conflicted: an analysis of conflicts of interest reported at the annual meetings of the Orthopaedic Trauma Association.

PURPOSE: To identify trends in industry sponsorship of orthopaedic trauma research presented at the annual meetings of the Orthopaedic Trauma Association since the establishment of conflict of interest (COI) reporting policies in 1993. BACKGROUND: Industry plays a large role in funding orthopaedic basic science and clinical research. The purpose of this study was to analyze the role of industrial support in orthopaedic research as documented in the final programs of the annual meetings of the Orthopaedic Trauma Association (OTA), determine the incidence and nature of COI in the papers and posters accepted for OTA presentation, and report any changes in the frequency of reporting since disclosure policies were enacted in 1993. METHODS: This paper analyzes COI for all years since the adoption of the reporting policies 1993-2002. From 1993-1998, presenters of posters and papers presented at the Orthopaedic Trauma Association annual meetings were required to disclose COI greater than dollar 500, the type of monetary distribution was not recorded. From 1999-2002, presenters of posters and papers were required to acknowledge the type of COI: 1. research grant, 2. miscellaneous non-income support, 3. royalties, 4. stock, and 5. consultant fees. All COI categories were recorded for each year Linear regression was used to determine significance of trends in the pooled data. RESULTS: There was an increase in the percentage of papers accepted and presented at the OTA between 1993 and 2002 with COI. The number of papers reporting COI rose from 7.6% in 1993 to 12.6% in 2002 (p = 0.0129). There was no significant increase in posters with COI over that same time period. No changes were observed in the nature of industrial involvement since the change in reporting enacted in 1999. There were no observed trends in NIH or OTA grant distribution between 1993 and 2002. DISCUSSION AND CONCLUSION: Industry is playing an increasing role in the funding oforthopaedic research. The majority of industrial support is in the form of research grants. The increasing industrial support of scientific research in the public sector is to be applauded as long as it does not lead to the sequestering and suppression of information that may be disadvantageous to the industrial sponsor.

Stress radiographs after ankle fracture: the effect of ankle position and deltoid ligament status on medial clear space measurements.

OBJECTIVE: This study was designed to determine 1) how ankle position affects the medial clear space by using stress radiographs, 2) which medial clear space measurement, overall width or increase in width, better predicts deep deltoid ligament disruption after Weber type-B distal fibular fracture, and 3) what value of medial clear space is most predictive of deep deltoid ligament disruption after Weber type-B distal fibular fracture. DESIGN: Cadaveric fracture model. SETTING: Biomechanics laboratory. INTERVENTION: Fluoroscopic mortise views were taken of 6 fresh cadaveric ankles mounted in a fixture permitting both positioning in neutral flexion, dorsiflexion, and plantarflexion, and the application of internal and external rotational forces. After destabilizing the ankles according to the supination-external rotation mechanism of Lauge-Hansen, repeat radiographs were taken with the same combination of flexion and applied rotational stress. MAIN OUTCOME MEASURE: Radiographic measurements of medial clear space width and changes in medial clear space were made. RESULTS: A medial clear space of > or =5 mm on radiographs taken in dorsiflexion with an external rotational stress was most predictive of deep deltoid ligament transection after distal fibular fracture. In dorsiflexion-external rotation, medial clear spaces of > or =4 mm yielded lower specificity and positive predictive value, whereas > or =6 mm yielded lower sensitivity and negative predictive value. All other stress conditions and increases in medial clear space of 2 or 3 mm were less predictive. CONCLUSIONS: Ankle stress radiographs taken in dorsiflexion-external rotation were most predictive of deep deltoid ligament disruption after distal fibular fracture. Under this stress condition, a medial clear space of > or =5 mm was the most reliable predictor of deep deltoid ligament status.

Management of extensor mechanism disruptions occurring after total knee arthroplasty.

Extensor mechanism disruption from quadriceps tendon rupture, patellar fracture, or patellar tendon rupture is an uncommon complication of total knee arthroplasty. Extensor mechanism disruption can occur either intraoperatively or postoperatively. Common intraoperative causes include avulsion or tendon injury arising from excessive tension during surgical exposure, improper patellar resection, and devascularization due to injudicious lateral retinacular release or multiple prior surgeries. The usual postoperative causes are tissue necrosis arising from infection, component malalignment, and trauma. A wide range of treatment options is available for managing these difficult problems, and recent advances in alternative techniques for reconstruction have yielded promising results.

Intramedullary fixation of unstable intertrochanteric hip fractures: one or two lag screws.

OBJECTIVE: To compare the screw sliding characteristics and biomechanical stability of four-part intertrochanteric hip fractures stabilized with an intramedullary nail using either one large-diameter lag screw (intramedullary hip screw [IMHS]; Smith & Nephew, Memphis, TN) or two small-diameter lag screws (trochanteric antegrade nail [TAN]; Smith & Nephew, Memphis, TN). DESIGN: Laboratory investigation using eight matched pairs of cadaveric human femurs with simulated, unstable intertrochanteric hip fractures. INTERVENTION: One femur of each matched pair was stabilized with an IMHS intramedullary nail, and the other was stabilized with a TAN intramedullary nail. Femurs were statically, then cyclically loaded on a servohydraulic materials testing machine. Finally, all specimens were loaded to failure. MAIN OUTCOME MEASURES: Screw sliding and inferior and lateral head displacements were measured for applied static loads from 500N to 1250N. The same measurements were obtained before and after cyclically loading the specimens at 1250N. Ultimate failure strength of the implant constructs also was determined. RESULTS: There was no significant difference between the TAN and IMHS in static or cyclical loading with respect to screw sliding or inferior and lateral head displacements. There was a statistically significant difference (P < 0.02) in failure strength, with the IMHS construct failing at an average of 2162N and the TAN construct failing at an average of 3238N. CONCLUSION: The two constructs showed equivalent rigidity and stability in all parameters assessed in elastic and cyclical tests. The TAN had a greater ultimate failure load.

Interobserver reliability and intraobserver reproducibility in suprascapular notch typing.

The size and shape of the suprascapular notch may be a factor in suprascapular nerve entrapment. The suprascapular notches of 623 scapulae were digitally photographed and used to determine notch type and area. Three researchers used to different classification systems for suprascapular notch typing. These systems were compared for interobserver reliability and intraobserver reproducibility using the kappa test. The mean kappa value for the classification used by Rengachary and colleagues was 0.468 and for the classification used by Ticker and associates was 0.531 for the inferior border of the notch and 0.736 for the superior border of the notch. The classification system used by Ticker and associates was more reliable and reproducible and produced both a superior and an inferior classification, making it possibly more clinically relevant than the classification system used by Rengachary and colleagues.

The shoulder in baseball pitching: biomechanics and related injuries-part 1.

The extreme range of motion at the shoulder, the high angular velocities and torques, and the repetitious nature of the pitching motion combine to make the shoulder vulnerable to injury during the baseball pitch. An understanding of the biomechanics that contribute to shoulder injuries during each phase of the pitching motion can facilitate the athlete's diagnosis, treatment, and rehabilitation. Common injuries that occur during the late cocking and acceleration phases of the pitch include anterior instability and impingement, bicipital tendinitis, and subacromial impingement. Nonoperative treatment consisting of an initial period of rest and NSAIDS, followed by physical therapy and a gradual return to activity, is usually successful. When this approach fails, surgical intervention, either arthroscopic or open, may be necessary. Physical therapy and rehabilitation are directed toward restoring the integrity and strength of the dynamic and static stabilizers of the shoulder joint, yet preserving the range of motion necessary for performance. Through rehabilitation, the dedicated athlete can often return to the pitching mound at his previous level of performance.

The shoulder in baseball pitching: biomechanics and related injuries-part 2.

The extreme range of motion at the shoulder, the high angular velocities and torques, and the repetitious nature of the pitching motion combine to make the shoulder vulnerable to injury during the baseball pitch. An understanding of the biomechanics that contribute to shoulder injuries during each phase of the pitching motion can facilitate the athlete's diagnosis, treatment, and rehabilitation. The athlete's symptoms and signs, as well as radiographic imaging, are key elements in arriving at a diagnosis of shoulder injuries. Nonoperative treatment consisting of an initial period of rest and NSAIDS, followed by physical therapy and a gradual return to activity, is usually successful. When this approach fails, surgical intervention, either arthroscopic or open, may be necessary. Physical therapy and rehabilitation are directed toward restoring the integrity and strength of the dynamic and static stabilizers of the shoulder joint, yet preserving the range of motion necessary for performance. Through rehabilitation, the dedicated athlete can often return to the pitching mound at his previous level of performance.