Faculty Bibliography

Long non-coding RNAs (lncRNAs), once considered 'genomic junk', have been found to regulate diverse biological processes and their study continues to reveal novel insights into lncRNA functions. Recent studies revealed that some lncRNAs may harbor small open reading frames (ORFs) that code for functional micropeptides. While investigating an unannotated primate-specific lncRNA, lncVLDLR, that is altered in patients with type II diabetes and cardiovascular disease, we discovered a previously unrecognized ORF encoding a 44 amino acid micropeptide. In vitro transcription and translation of the IMP coding sequence in the presence of ³⁵S-methionine produced a single 8 kDa peptide, which we have named Inflammation-modulating MicroPeptide (IMP). To dissect IMP function, we focused on its amino acid sequence and putative structure. These analyses revealed high sequence homology between IMP and transcription factors such as NFKB, c-myb and zinc finger proteins, and the presence of a hydrophobic region with an LxxLL motif often found in transcriptional regulators. Circular dichroism spectroscopy of synthesized IMP predicted an intrinsically disordered peptide, which is a common characteristic of transcriptional coactivators. To investigate a potential role of IMP in regulating gene transcription, we cloned a MYC-epitope tag in-frame with IMP within the full-length transcript of lncVLDLR and expressed it in HEK293 cells. Immunofluorescence staining, and cell fractionation combined with western blotting, confirmed nuclear localization of IMP RNA-seq analysis of THP1 macrophages overexpressing IMP revealed an increase in inflammatory genes, including cytokines and chemokines. Moreover, analysis of upstream regulators of these genes suggests that IMP may interact with KIX domaincontaining transcriptional coactivators to regulate inflammatory gene expression. Together our data identify a novel human micropeptide, encoded within a putative lncRNA that is dysregulated in diabetes and cardiovascular disease, that regulates inflammatory gene transcription. Further characterization of IMP and its regulatory network may uncover novel opportunities for therapeutic intervention in cardiovascular and other inflammatory diseases

Seminal yeast studies have established the value of comprehensively mapping genetic interactions (GIs) for inferring gene function. Efforts in human cells using focused gene sets underscore the utility of this approach, but the feasibility of generating large-scale, diverse human GI maps remains unresolved. We developed a CRISPR interference platform for large-scale quantitative mapping of human GIs. We systematically perturbed 222,784 gene pairs in two cancer cell lines. The resultant maps cluster functionally related genes, assigning function to poorly characterized genes, including TMEM261, a new electron transport chain component. Individual GIs pinpoint unexpected relationships between pathways, exemplified by a specific cholesterol biosynthesis intermediate whose accumulation induces deoxynucleotide depletion, causing replicative DNA damage and a synthetic-lethal interaction with the ATR/9-1-1 DNA repair pathway. Our map provides a broad resource, establishes GI maps as a high-resolution tool for dissecting gene function, and serves as a blueprint for mapping the genetic landscape of human cells.

The use of temporomandibular joint (TMJ) implants is considered to be a reliable treatment for some TMJ disorders when TMJ anatomical integrity is compromised. Among all of the designs proposed for these devices, intramedullary approaches are relatively new, and they may offer several advantages compared to those of past models with a lateral approach. In this report, we use finite element analysis (FEA) to calculate stress forces of a TMJ implant featuring a ferrule ring, which is frequently used in engineering as a stress distractor to reduce the splinter effect. Our analysis suggests that the addition of a ferrule ring in the TMJ implant helps to reduce von Mises stresses in the device and displacement forces in the volume and surface of the implant. These results suggest that including a ferrule ring in a TMJ implant may contribute to the stability and outcome of a TMJ implant by reducing component stress and displacement forces.

Influenza A virus is an enveloped virus with a segmented genome consisting of eight negative-sense, single-stranded RNAs. Accumulating evidence has revealed that influenza viruses selectively package their genomes. However, less is known about how different viral RNA segments are selected for incorporation into progeny virions. Understanding the trafficking routes and assembly process of various viral RNA segments during infection will shed light on the mechanisms of selective genome packaging for influenza A viruses. This chapter describes the single-molecule sensitivity RNA fluorescence in situ hybridization assay (smFISH) for influenza viral RNAs, a method used to analyze the distributions and trafficking of viral RNAs in infected cells with segment specificity. Hybridization using 20 or more short fluorescently labeled DNA probes allows the detection of viral RNAs with single-molecule sensitivity. The following imaging analyses provide information regarding quantitative measurements of vRNA abundance and the relative positions of the different viral RNA segments in cells. This chapter also includes a protocol for combining immunofluorescence techniques with smFISH, which is useful to analyze the positions of viral RNAs relative to viral/cellular proteins in infected cells.

Background: Although much clinical progress has been made in harnessing the immune system to recognize and target cancer, there is still a significant lack of an understanding of how tumors evade immune recognition and the mechanisms that drive tumor resistance to both T cell and checkpoint blockade immunotherapy. Our objective is to understand how tumor-mediated signaling through inhibitory receptors, including PD-1, combine to affect the process of T cell recognition of tumor antigen and activation signaling, with the goal of understanding the basis of resistance to PD-1 blockade and the potential identification of new molecular targets to enable T cells to overcome dysfunction mediated by multiple inhibitory receptors.
Methods and Results: We show that Lck activity affects T cell sensitivity and influences the probability of inducing effector function [1]. Under non-activating conditions, Lck and Shp-1 phosphorylation and activity vary based on CD8+ memory T cell phenotype. Shp-1 interaction with Lck under non-activation conditions can also vary, as suggested by our results showing decreased Shp-1 S591 phosphorylation, which affects Shp-1 localization and correlates with increased Shp-1 colocalization with Lck. Further, we showed that Shp-1 directly influences Lck activity under non-activating conditions, as inhibition of Shp-1 leads to increased Lck activity. Importantly, inhibition of Shp- 1/2, a major mediator of PD-1 signaling, targeting CD28 and Lck [2], prior to activation leads to increased T cell cytotoxic effector function. Our proteomics-based analysis of patient T cells identified both mediators of PD-1 signaling and signaling components and pathways associated with blockade resistance. It has generally been thought that TCR and CD8 binding depend mainly on their ectodomain interactions with pMHC. We have shown, however, that Lck-CD8 binding [3] and Lck activity [4] are required for upregulated CD8 binding to prebound TCR-pMHC complex. Therefore, the cytoplasmic associations of Lck with CD8 and Zap-70, as well as CD3 with Zap-70 may influence formation and stability of the TCRpMHCCD8 complex. To determine the mechanistic basis of PD-1 inhibition of TCR-pMHCCD8 binding we utilized 2D affinity combined with Biomembrane Force Probe (BFP) measurements[5, 6] and showed that PD-1 directly suppresses TCR pMHCCD8 binding. Our data also revealed that TCR-pMHC binding was independent of PD-1-PD-L1, but TCRpMHCCD8 binding was suppressed by PD-1-PD-L1 binding demonstrating negative cooperativity, as fewer bonds formed than the sum of bonds formed by each interaction alone.
Conclusion(s): Together, our results show that the activities of TCRproximal signaling components affect T cell mechanosensing and sensitivity at the earliest stages of antigen recognition and are influenced by PD-1. Targeting these interactions may enhance tumor-specific T cell sensitivity for cancer immunotherapy and understanding the basis of resistance to PD-1 blockade to potentially allow identification of new molecular targets to enable T cells to overcome dysfunction mediated by multiple inhibitory receptors

In the version of this Article originally published, the surname of the author Edward A. Fisher was spelt incorrectly as 'Fischer'. This has now been corrected.

Cryo electron microscopy facilities running multiple instruments and serving users with varying skill levels need a robust and reliable method for benchmarking both the hardware and software components of their single particle analysis workflow. The workflow is complex, with many bottlenecks existing at the specimen preparation, data collection and image analysis steps; the samples and grid preparation can be of unpredictable quality, there are many different protocols for microscope and camera settings, and there is a myriad of software programs for analysis that can depend on dozens of settings chosen by the user. For this reason, we believe it is important to benchmark the entire workflow, using a standard sample and standard operating procedures, on a regular basis. This provides confidence that all aspects of the pipeline are capable of producing maps to high resolution. Here we describe benchmarking procedures using a test sample, rabbit muscle aldolase.

Single particle cryo-electron microscopy (cryoEM) is often performed under the assumption that particles are not adsorbed to the air-water interfaces and in thin, vitreous ice. In this study, we performed fiducial-less tomography on over 50 different cryoEM grid/sample preparations to determine the particle distribution within the ice and the overall geometry of the ice in grid holes. Surprisingly, by studying particles in holes in 3D from over 1000 tomograms, we have determined that the vast majority of particles (approximately 90%) are adsorbed to an air-water interface. The implications of this observation are wide-ranging, with potential ramifications regarding protein denaturation, conformational change, and preferred orientation. We also show that fiducial-less cryo-electron tomography on single particle grids may be used to determine ice thickness, optimal single particle collection areas and strategies, particle heterogeneity, and de novo models for template picking and single particle alignment.

We present an update describing new features and applications of Spotiton, a novel instrument for vitrifying samples for cryoEM. We have used Spotiton to prepare several test specimens that can be reconstructed using routine single particle analysis to ∼3 Å resolution, indicating that the process has no apparent deleterious effect on the sample integrity. The system is now in routine and continuous use in our lab and has been used to successfully vitrify a wide variety of samples.

In Figs. 2b and 3d of this Letter, the labels 'Dynamin 1' and 'Overlay' were inadvertently swapped. This has been corrected online.