High-density resolution of the Kaposi's sarcoma associated herpesvirus transcriptome identifies novel transcript isoforms generated by long-range transcription and alternative splicing.

Kaposi's sarcoma-associated herpesvirus is the etiologic agent of Kaposi's sarcoma and two B-cell malignancies. Recent advancements in sequencing technologies have led to high resolution transcriptomes for several human herpesviruses that densely encode genes on both strands. However, for KSHV progress remained limited due to the overall low percentage of KSHV transcripts, even during lytic replication. To address this challenge, we have developed a target enrichment method to increase the KSHV-specific reads for both short- and long-read sequencing platforms. Furthermore, we combined this approach with the Transcriptome Resolution through Integration of Multi-platform Data (TRIMD) pipeline developed previously to annotate transcript structures. TRIMD first builds a scaffold based on long-read sequencing and validates each transcript feature with supporting evidence from Illumina RNA-Seq and deepCAGE sequencing data. Our stringent innovative approach identified 994 unique KSHV transcripts, thus providing the first high-density KSHV lytic transcriptome. We describe a plethora of novel coding and non-coding KSHV transcript isoforms with alternative untranslated regions, splice junctions and open-reading frames, thus providing deeper insights on gene expression regulation of KSHV. Interestingly, as described for Epstein-Barr virus, we identified transcription start sites that augment long-range transcription and may increase the number of latency-associated genes potentially expressed in KS tumors.

The microRNAs miR-449a and miR-424 suppress osteosarcoma by targeting cyclin A2 expression.

MicroRNAs of the miR-16 and miR-34 families have been reported to inhibit cell cycle progression, and their loss has been linked to oncogenic transformation. Utilizing a high-throughput, genome-wide screen for miRNAs and mRNAs that are differentially regulated in osteosarcoma (OS) cell lines, we report that miR-449a and miR-424, belonging to the miR-34 and miR-16 families, respectively, target the major S/G2 phase cyclin, cyclin A2 (CCNA2), in a bipartite manner. We found that the 3'-UTR of CCNA2 is recognized by miR-449a, whereas the CCNA2 coding region is targeted by miR-424. Of note, we observed loss of both miR-449a and miR-424 in OS, resulting in derepression of CCNA2 and appearance of aggressive cancer phenotypes. Ectopic expression of miR-449a and miR-424 significantly decreased cyclin A2 levels and inhibited proliferation rate, migratory potential, and colony-forming ability of OS cells. To further probe the roles of miR-449a and miR-424 in OS, we developed an OS mouse model by intraosseous injection of U2OS cells into the tibia bone of NOD-scid mice, which indicated that miR-449a and miR-424 co-expression suppresses tumor growth. On the basis of this discovery, we analyzed the gene expression of human OS biopsy samples, revealing that miR-449a and miR-424 are both down-regulated, whereas cyclin A2 is significantly up-regulated in these OS samples. In summary, the findings in our study highlight that cyclin A2 repression by miRNAs of the miR-16 and miR-34 families is lost in aggressive OS.

MicroRNA-874-mediated inhibition of the major G1/S phase cyclin, CCNE1, is lost in osteosarcomas.

The tumor microenvironment is characterized by nutrient-deprived conditions in which the cancer cells have to adapt for survival. Serum starvation resembles the growth factor deprivation characteristic of the poorly vascularized tumor microenvironment and has aided in the discovery of key growth regulatory genes and microRNAs (miRNAs) that have a role in the oncogenic transformation. We report here that miR-874 down-regulates the major G1/S phase cyclin, cyclin E1 (CCNE1), during serum starvation. Because the adaptation of cancer cells to the tumor microenvironment is vital for subsequent oncogenesis, we tested for miR-874 and CCNE1 interdependence in osteosarcoma cells. We observed that miR-874 inhibits CCNE1 expression in primary osteoblasts, but in aggressive osteosarcomas, miR-874 is down-regulated, leading to elevated CCNE1 expression and appearance of cancer-associated phenotypes. We established that loss of miR-874-mediated control of cyclin E1 is a general feature of osteosarcomas. The down-regulation of CCNE1 by miR-874 is independent of E2F transcription factors. Restoration of miR-874 expression impeded S phase progression, suppressing aggressive growth phenotypes, such as cell invasion, migration, and xenograft tumors, in nude mice. In summary, we report that miR-874 inhibits CCNE1 expression during growth factor deprivation and that miR-874 down-regulation in osteosarcomas leads to CCNE1 up-regulation and more aggressive growth phenotypes.

RPA70 depletion induces hSSB1/2-INTS3 complex to initiate ATR signaling.

The primary eukaryotic single-stranded DNA-binding protein, Replication protein A (RPA), binds to single-stranded DNA at the sites of DNA damage and recruits the apical checkpoint kinase, ATR via its partner protein, ATRIP. It has been demonstrated that absence of RPA incapacitates the ATR-mediated checkpoint response. We report that in the absence of RPA, human single-stranded DNA-binding protein 1 (hSSB1) and its partner protein INTS3 form sub-nuclear foci, associate with the ATR-ATRIP complex and recruit it to the sites of genomic stress. The ATRIP foci formed after RPA depletion are abrogated in the absence of INTS3, establishing that hSSB-INTS3 complex recruits the ATR-ATRIP checkpoint complex to the sites of genomic stress. Depletion of homologs hSSB1/2 and INTS3 in RPA-deficient cells attenuates Chk1 phosphorylation, indicating that the cells are debilitated in responding to stress. We have identified that TopBP1 and the Rad9-Rad1-Hus1 complex are essential for the alternate mode of ATR activation. In summation, we report that the single-stranded DNA-binding protein complex, hSSB1/2-INTS3 can recruit the checkpoint complex to initiate ATR signaling.

The crosstalk between miRNAs and signaling pathways in human cancers: Potential therapeutic implications.

MicroRNAs (miRNAs) are increasingly recognized as central players in the regulation of eukaryotic physiological processes. These small double stranded RNA molecules have emerged as pivotal regulators in the intricate network of cellular signaling pathways, playing significant roles in the development and progression of human cancers. The central theme in miRNA-mediated regulation of signaling pathways involves their ability to target and modulate the expression of pathway components. Aberrant expression of miRNAs can either promote or suppress key signaling events, influencing critical cellular processes such as proliferation, apoptosis, angiogenesis, and metastasis. For example, oncogenic miRNAs often promote cancer progression by targeting tumor suppressors or negative regulators of signaling pathways, thereby enhancing pathway activity. Conversely, tumor-suppressive miRNAs frequently inhibit oncogenic signaling by targeting key components within these pathways. This complex regulatory crosstalk underscores the significance of miRNAs as central players in shaping the signaling landscape of cancer cells. Furthermore, the therapeutic implications of targeting miRNAs in cancer are substantial. miRNAs can be manipulated to restore normal signaling pathway activity, offering a potential avenue for precision medicine. The development of miRNA-based therapeutics, including synthetic miRNA mimics and miRNA inhibitors, has shown promise in preclinical and clinical studies. These strategies aim to either enhance the activity of tumor-suppressive miRNAs or inhibit the function of oncogenic miRNAs, thereby restoring balanced signaling and impeding cancer progression. In conclusion, the crosstalk between miRNAs and signaling pathways in human cancers is a dynamic and influential aspect of cancer biology. Understanding this interplay provides valuable insights into cancer development and progression. Harnessing the therapeutic potential of miRNAs as regulators of signaling pathways opens up exciting opportunities for the development of innovative cancer treatments with the potential to improve patient outcomes. In this chapter, we provide an overview of the crosstalk between miRNAs and signaling pathways in the context of cancer and highlight the potential therapeutic implications of targeting this regulatory interplay.

From Detection to Protection: Antibodies and Their Crucial Role in Diagnosing and Combatting SARS-CoV-2.

Understanding the antibody response to SARS-CoV-2, the virus responsible for COVID-19, is crucial to comprehending disease progression and the significance of vaccine and therapeutic development. The emergence of highly contagious variants poses a significant challenge to humoral immunity, underscoring the necessity of grasping the intricacies of specific antibodies. This review emphasizes the pivotal role of antibodies in shaping immune responses and their implications for diagnosing, preventing, and treating SARS-CoV-2 infection. It delves into the kinetics and characteristics of the antibody response to SARS-CoV-2 and explores current antibody-based diagnostics, discussing their strengths, clinical utility, and limitations. Furthermore, we underscore the therapeutic potential of SARS-CoV-2-specific antibodies, discussing various antibody-based therapies such as monoclonal antibodies, polyclonal antibodies, anti-cytokines, convalescent plasma, and hyperimmunoglobulin-based therapies. Moreover, we offer insights into antibody responses to SARS-CoV-2 vaccines, emphasizing the significance of neutralizing antibodies in order to confer immunity to SARS-CoV-2, along with emerging variants of concern (VOCs) and circulating Omicron subvariants. We also highlight challenges in the field, such as the risks of antibody-dependent enhancement (ADE) for SARS-CoV-2 antibodies, and shed light on the challenges associated with the original antigenic sin (OAS) effect and long COVID. Overall, this review intends to provide valuable insights, which are crucial to advancing sensitive diagnostic tools, identifying efficient antibody-based therapeutics, and developing effective vaccines to combat the evolving threat of SARS-CoV-2 variants on a global scale.

DExD/H Box Helicases DDX24 and DDX49 Inhibit Reactivation of Kaposi's Sarcoma Associated Herpesvirus by Interacting with Viral mRNAs.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic gammaherpesvirus that is the causative agent of primary effusion lymphoma and Kaposi's sarcoma. In healthy carriers, KSHV remains latent, but a compromised immune system can lead to lytic viral replication that increases the probability of tumorigenesis. RIG-I-like receptors (RLRs) are members of the DExD/H box helicase family of RNA binding proteins that recognize KSHV to stimulate the immune system and prevent reactivation from latency. To determine if other DExD/H box helicases can affect KSHV lytic reactivation, we performed a knock-down screen that revealed DHX29-dependent activities appear to support viral replication but, in contrast, DDX24 and DDX49 have antiviral activity. When DDX24 or DDX49 are overexpressed in BCBL-1 cells, transcription of all lytic viral genes and genome replication were significantly reduced. RNA immunoprecipitation of tagged DDX24 and DDX49 followed by next-generation sequencing revealed that the helicases bind to mostly immediate-early and early KSHV mRNAs. Transfection of expression plasmids of candidate KSHV transcripts, identified from RNA pull-down, demonstrated that KSHV mRNAs stimulate type I interferon (alpha/beta) production and affect the expression of multiple interferon-stimulated genes. Our findings reveal that host DExD/H box helicases DDX24 and DDX49 recognize gammaherpesvirus transcripts and convey an antiviral effect in the context of lytic reactivation.