Zhi-Ming Zheng

SRSF3 (SRp20) is an essential RNA splicing factor. By conditional knockout (KO) of Srsf3, we found Srsf3 at its physiological level plays a tumor-suppressive role in mouse liver carcinogenesis. Srsf3 KO in hepatocytes disrupted sex disparity in the development of DEN-induced liver cancer and resulted in high incidence of liver cancer in both male and female mice. In contrast, Srsf3 is oncogenic in the mouse mammary glands and Srsf3 KO significantly delayed the development of Erbb2 breast cancer. Srsf3 KO significantly increased the expression of transcription factors Sox4, E2f1, and Myc, calcium channel factor Trpv4, and zinc-binding Trim6 in liver cancer, but did not in Erbb2 breast cancer. Srsf3 KO enhanced the expression of glucose transmembrane transporter Mfsd4a and translation initiator Eif4a2 in DEN-induced liver cancer but reduced Mfsd4a and Eif4a2 expression in breast cancer. More importantly, Srsf3 KO reduced the expression of hepatocyte ERα and transcription factor Foxa family genes essential for sexual dimorphism in liver cancer and their downstream targets leukemia inhibitory factor receptor Lifr and epidermal growth factor receptor Egfr, but increased the expression of Myc. We further evidenced that SRSF3 binds to FOXA1 promoter and is responsible for FOXA1 promoter activity and FOXA1 expression in liver cancer cells. Together, our data highlight a functional paradigm of SRSF3 at its physiological level in tissue context-dependent gene regulation and in facilitating hepatocyte ERα and FOXA1 expression for sex disparity in liver cancer development.

Kaposi's sarcoma‐associated herpesvirus (KSHV) ORF57, also known as MTA, is a viral nonstructural protein that belongs to a conserved family of functionally related proteins encoded by all herpesviruses. As an RNA‐binding protein, ORF57/MTA functions as a key post‐transcriptional regulator of viral gene expression and is essential for efficient viral replication and production of infectious virions. ORF57/MTA features two distinguishable domains: an intrinsic disordered N‐terminal domain or NTD for protein‐protein and protein‐RNA interactions and a structural α‐helix‐rich C‐terminal domain or CTD for ORF57/MTA dimerization to prevent its proteasome degradation. ORF57/MTA enhances viral gene expression by promoting RNA stability, RNA splicing, and translation of viral transcripts through its NTD domain in cooperation with host/viral cofactors as distinct individual ribonucleoprotein complexes. To mediate these diverse activities, ORF57/MTA binds MTA‐responsive elements (MREs) within target RNAs in a sequence and structural motif‐dependent manner. Most recent progresses in this update since 2015 include determination of ORF57/MTA domain structure and the crystal structure of its CTD, discovery of a novel role of ORF57/MTA in counteracting cellular innate immune responses through modulation of RNA granule biogenesis, genome‐wide identification of viral and host RNAs as ORF57/MTA targets, and elucidation of long‐distance viral RNA splicing crossing a large part of the KSHV genome. Collectively, these findings represent significant steps toward our understanding of ORF57/MTA‐mediated regulation of not only viral but also host gene expression, as exemplified by ORF57/MTA regulation of FOS expression. Nevertheless, many aspects of ORF57/MTA function remain to be determined and warrant future investigation.

In coronavirus (CoV)-infected cells, several structural and accessory proteins are synthesized from subgenome RNAs (sgRNA) containing a common genomic 5ʹ-leader followed by a given open reading frame (ORF). We report that the abundance of these sgRNAs varies with distance from the 3′-end of the genome. Thus, there are more sgRNAs encoding nucleocapsid (N) than spike (S), presumably the results from discontinuous 5ʹ−3ʹ transcription template switch mediated by the viral replication and transcription complex (RTC). We optimized the circular polymerase extension reaction (CPER) methodology to generate infectious double-stranded circular cDNA (ds-circDNA) containing the mNeonGreen (NG) reporter in accessory ORFs of human CoVs OC43 and SARS-CoV-2. In each CoV, we found that levels of sgRNAs and NG expression increased with 3ʹ proximal genomic NG location. By reinfection of HCT-8 cells with the same MOI 0.01, however, we found that the slow-growing OC43 NG-ns2 virions exhibited equal infectivity and productivity as the fast-growing OC43 NG-ns12.9 virions. Introduction of point-mutations into the mapped TRS _B motif for synthesis of the OC43 ns12.9 and M sgRNAs led to disrupt TRS _B -TRS _L cross-interactions and block production of the corresponding sgRNAs and infectious virions. Together, our data indicate that, by using an optimized CPER approach for positional NG insertions, we demonstrated the progressional reduction of the RTC-mediated template switch (read-through individual TRS _B ) efficiency in production of the corresponding sgRNAs in an order from the 3ʹ viral genome end. IMPORTANCE: The mechanism for sgRNA synthesis in the coronavirus life cycle is poorly understood. The current model suggests discontinuous template-switch transcription mediated by viral replication and transcription complex (RTC) for synthesis of individual sgRNAs to translate corresponding structural and accessory proteins but lacks experimental data support. This report provides the first experimental evidence that, in both hCoV-OC43 and SARS-CoV-2, viral RTC synthesizes its sgRNAs by long-range base-pairing between a distal transcription regulatory body sequence (TRS _B ) upstream of each structural/accessory ORF and the transcription regulatory leader sequence (TRS _L ) from the viral genome 5ʹ-UTR, leading to the production of viral sgRNAs in abundance order from the viral genome 3ʹ -end, with more N sgRNAs but less S sgRNAs. Our data support a “first-come, first-serving” model in TRS _B -TRS _L cross-interaction and read-through TRS _B process to mediate discontinuous transcription switch in coronavirus sgRNA synthesis in a 5ʹ−3ʹ transcription direction from the 3ʹ viral genome during coronavirus infection.

Mouse papillomavirus (MmuPV1) is the first papillomavirus known to infect laboratory mice, making it an irreplaceable tool for research on papillomaviruses. Despite wide use, standardized techniques for conducting MmuPV1 animal research are lacking. In this report, we describe an unexpected MmuPV1 outbreak causing recurrent papillomatosis in a specific pathogen-free animal research facility. The infected mice displayed characteristic papillomatosis lesions from the muzzles, tails, and feet with histological signs including anisocytosis, epithelial dysplasia, and typical koilocytosis. Etiology studies showed that the papilloma tissues exhibited MmuPV1 infection with expression of viral early and late genes detected by RNA-ISH using MmuPV1 antisense probe to viral E6E7 region and antisense probe to viral L1 region. The viral L1 protein was detected by an anti-MmuPV1 L1 antibody. PCR amplification and cloning of the entire viral genome showed that the origin of the outbreak virus, named MmuPV1 Bethesda strain (GenBank Acc. No. PX123224), could be traced to the MmuPV1 virus previously used in studies at the same facility. Our data indicate that MmuPV1 could exist in a contaminated environment for a long period of time, and a standardized international animal protocol discussing how to handle MmuPV1 studies is urgently needed.

Increased expression of lnc-FANCI-2, a newly discovered long noncoding RNA, is associated with cervical lesion progression from cervical intraepithelial neoplasia stage 1 (CIN1, low grade), CIN2–3 (high grade), to cervical cancer. Viral oncoprotein E7 of high-risk human papillomaviruses (HR-HPVs) and host transcription factor YY1 are two major factors promoting lnc-FANCI-2 expression. Using CRISPR-Cas9 technology, we knocked out the expression of lnc-FANCI-2 in the HPV16-positive cervical cancer cell line, CaSki cells. The selected knockout (KO) single-cell clones displayed altered cell morphology and proliferation with changes of cellular soluble receptors, but normal HPV16 E6 and E7 expression. Relative to the parental cells, lnc-FANCI-2 KO cells exhibited significantly increased RAS signaling and epithelial-mesenchymal transition, but decreased response to IFN signaling, along with increased p-Akt and p-Erk1/2 (two RAS signaling effectors), IGFBP3, MCAM, VIM, and CCND2 (cyclin D2) and decreased expression of RAC3. lnc-FANCI-2 in CaSki interacts with cellular proteins H13, HNRH1, K1H1, MAP4K4, and RNPS1. MAP4K4 knockdown led to enhance the expression of p-Erk1/2 and p-Akt. High lnc-FANCI-2 and low MCAM levels in cervical cancer tissues were found to be associated with patients’ survival. A key function of lnc-FANCI-2 intrinsically regulates RAS signaling to impact cervical lesion progression and cervical cancer prognosis.

We recently discovered the expression of a long noncoding RNA, lnc-FANCI-2, coinciding with cervical lesion progression from CIN1, CIN2-3 to cervical cancer. Viral E7 of high-risk HPVs and host transcription factor YY1 are two major factors promoting lnc-FANCI-2 expression. To explore possible roles of lnc-FANCI-2 in HPV-induced cervical carcinogenesis, we ablated the expression of lnc-FANCI-2 in the HPV16-positive cervical cancer cell line, CaSki. Knock-out (KO) single cell clones expressed HPV16 oncogenes normally but displayed altered cell morphology and proliferation when compared with the parental cells. Proteomic profiling of cytosolic and secreted proteins from the parental and KO cells showed that lnc-FANCI-2 regulates expression of a subset of cell soluble receptors responsible for cell signaling, epithelial mesenchymal transition, and IFN responses. RNA-seq analyses revealed that, relative to the parental cells, lnc-FANCI-2 KO cells exhibited significantly increased RAS signaling but decreased IFN pathways. In the KO cells, phosphorylated Akt and Erk1/2, two important RAS pathway effectors, were increased more than 3-fold, accompanied by increase of IGFBP3, MCAM, VIM, and CCND2 (cyclin D2) and decrease of RAC3 expression. High levels of lnc-FANCI-2 and lower levels of MCAM in cervical cancer patients are associated with improved survival. We found that lnc-FANCI-2 interacts specifically with 32 host proteins, including H13, HNRH1, K1H1, MAP4K4, and RNPS1, and knockdown of MAP4K4 in CaSki cells led to an increase of phosphorylation of Erk1/2 and somewhat Akt. In summary, a key function of lnc-FANCI-2 is to intrinsically regulate RAS signaling to impact cervical lesion progression and affect cervical cancer prognosis.

SRSF3 (SRp20) is an essential splicing factor. We discovered Srsf3 plays an oncogenic role in breast cancer and Srsf3 knockout (KO) in mammary glands delays the development of breast cancer in an Erbb2 mouse model. In contrast, Srsf3 is tumor-suppressive in mouse liver tissues. Srsf3 KO in hepatocytes enhances DEN-induced liver cancer and disrupts the sex disparity in DEN-induced liver cancer. Comparing to Srsf3 WT liver cancer, Srsf3 KO significantly increases Sox4, E2f1, Trpv4, Trim6, and Myc expression, but does not so in Erbb2 breast cancer. Srsf3 KO inhibits expression of Mfsd4a and Eif4a2 in breast cancer but enhances Mfsd4a and Eif4a2 expression in liver cancer. Moreover, Srsf3 KO suppresses the expression of ERα and Foxa genes to reduce Lifr and Egfr but induce Myc expression and promote liver cancer in female mice. Together, our data highlight a new functional paradigm of SRSF3 at its physiological level in tissue context-dependent gene regulation.

Kaposi’s sarcoma-associated herpesvirus (KSHV) encodes an RNA-binding protein ORF57 in lytic infection. Using an optimized CLIP-seq in this report, we identified ORF57-bound transcripts from 544 host protein-coding genes. By comparing with the RNA-seq profiles from BCBL-1 cells with latent and lytic KSHV infection and from HEK293T cells with and without ORF57 expression, we identified FOS RNA as one of the major ORF57-specific RNA targets. FOS dimerizes with JUN as a transcription factor AP-1 involved in cell proliferation, differentiation, and transformation. Knockout of the ORF57 gene from the KSHV genome led BAC16-iSLK cells incapable of FOS expression in KSHV lytic infection. The dysfunctional KSHV genome in FOS expression could be rescued by Lenti-ORF57 virus infection. ORF57 protein does not regulate FOS translation but binds to the 13-nt RNA motif near the FOS RNA 5′ end and prolongs FOS mRNA half-life 7.7 times longer than it is in the absence of ORF57. This binding of ORF57 to FOS RNA is likely competitive to the binding of host nuclease AEN (ISG20L1) of which physiological RNase activity remains unknown. KSHV infection inhibits the expression of AEN, but not exosomal RNA helicase MTR4. FOS expression mediated by ORF57 inhibits AEN transcription through FOS binding to AEN promoter but transactivates RGS2 , a regulator of G-protein-coupled receptors. FOS binds a conserved AP-1 site in the RGS2 promoter and enhances RGS2 expression to phosphorylate AKT. Altogether, we have discovered that KSHV ORF57 specifically binds and stabilizes FOS RNA to increase FOS expression, thereby disturbing host gene expression and inducing pathogenesis during KSHV lytic infection. IMPORTANCE: We discovered that FOS, a heterodimer component of oncogenic transcription factor AP-1, is highly elevated in KSHV-infected cells by expression of a viral lytic RNA-binding protein, ORF57, which binds a 13-nt RNA motif near the FOS RNA 5′ end to prolong FOS RNA half-life. This binding of ORF57 to FOS RNA is competitive to the binding of host RNA destabilizer(s). KSHV infection inhibits expression of host nuclease AEN, but not MTR4. FOS inhibits AEN transcription by binding to the AEN promoter but transactivates RGS2 by binding to a conserved AP-1 site in the RGS2 promoter, thereby enhancing RGS2 expression and phosphorylation of AKT. Thus, KSHV lytic infection controls the expression of a subset of genes for signaling, cell cycle progression, and proliferation to potentially contribute to viral oncogenesis.