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1.  Alternative RNA splicing of KSHV ORF57 produces two different RNA isoforms 
Virology  2015;488:81-87.
In lytically infected B cells Kaposi sarcoma-associated herpesvirus (KSHV) ORF57 gene encodes two RNA isoforms by alternative splicing of its pre-mRNA, which contains a small, constitutive intron in its 5′ half and a large, suboptimal intron in its 3’s half. The RNA1 isoform encodes full-length ORF57 and is a major isoform derived from splicing of the constitutive small intron, but retaining the suboptimal large intron as the coding region. A small fraction (<5%) of ORF57 RNA undergoes double splicing to produce a smaller non-coding RNA2 due to lack of a translational termination codon. Both RNAs are cleaved and polyadenylated at the same cleavage site CS83636. The insertion of ORF57 RNA1 into a restriction cutting site in certain mammalian expression vectors activates splicing of the subopitmal intron and produces a truncated ORF57 protein.
Graphical Abstract
doi:10.1016/j.virol.2015.10.031
PMCID: PMC4744540  PMID: 26609938
KSHV; ORF57; alternative splicing
2.  Stability of Structured Kaposi's Sarcoma-Associated Herpesvirus ORF57 Protein Is Regulated by Protein Phosphorylation and Homodimerization 
Journal of Virology  2015;89(6):3256-3274.
ABSTRACT
Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 plays an essential role in KSHV lytic infection by promoting viral gene expression at the posttranscriptional level. Using bioinformatic and biochemical approaches, we determined that ORF57 contains two structurally and functionally distinct domains: a disordered nonstructural N-terminal domain (amino acids [aa] 1 to 152) and a structured α-helix-rich C-terminal domain (aa 153 to 455). The N-terminal domain mediates ORF57 interaction with several RNA-protein complexes essential for ORF57 to function. The N-terminal phosphorylation by cellular casein kinase II (CKII) at S21, T32, and S43, and other cellular kinases at S95 and S97 residues in proximity of the caspase-7 cleavage site, 30-DETD-33, inhibits caspase-7 digestion of ORF57. The structured C-terminal domain mediates homodimerization of ORF57, and the critical region for this function was mapped carefully to α-helices 7 to 9. Introduction of point mutations into α-helix 7 at ORF57 aa 280 to 299, a region highly conserved among ORF57 homologues from other herpesviruses, inhibited ORF57 homodimerization and led to proteasome-mediated degradation of ORF57 protein. Thus, homodimerization of ORF57 via its C terminus prevents ORF57 from degrading and allows two structure-free N termini of the dimerized ORF57 to work coordinately for host factor interactions, leading to productive KSHV lytic infection and pathogenesis.
IMPORTANCE KSHV is a human oncogenic virus linked to the development of several malignancies. KSHV-mediated oncogenesis requires both latent and lytic infection. The KSHV ORF57 protein is essential for KSHV lytic replication, as it regulates the expression of viral lytic genes at the posttranscriptional level. This report provides evidence that the structural conformation of the ORF57 protein plays a critical role in regulation of ORF57 stability. Phosphorylation by CKII on the identified serine/threonine residues at the N-terminal unstructured domain of ORF57 prevents its digestion by caspase-7. The C-terminal domain of ORF57, which is rich in α-helices, contributes to homodimerization of ORF57 to prevent proteasome-mediated protein degradation. Elucidation of the ORF57 structure not only enables us to better understand ORF57 stability and functions but also provides an important tool for us to modulate ORF57's activity with the aim to inhibit KSHV lytic replication.
doi:10.1128/JVI.03721-14
PMCID: PMC4337553  PMID: 25568207
3.  E6^E7, a Novel Splice Isoform Protein of Human Papillomavirus 16, Stabilizes Viral E6 and E7 Oncoproteins via HSP90 and GRP78 
mBio  2015;6(1):e02068-14.
ABSTRACT
Transcripts of human papillomavirus 16 (HPV16) E6 and E7 oncogenes undergo alternative RNA splicing to produce multiple splice isoforms. However, the importance of these splice isoforms is poorly understood. Here we report a critical role of E6^E7, a novel isoform containing the 41 N-terminal amino acid (aa) residues of E6 and the 38 C-terminal aa residues of E7, in the regulation of E6 and E7 stability. Through mass spectrometric analysis, we identified that HSP90 and GRP78, which are frequently upregulated in cervical cancer tissues, are two E6^E7-interacting proteins responsible for the stability and function of E6^E7, E6, and E7. Although GRP78 and HSP90 do not bind each other, GRP78, but not HSP90, interacts with E6 and E7. E6^E7 protein, in addition to self-binding, interacts with E6 and E7 in the presence of GRP78 and HSP90, leading to the stabilization of E6 and E7 by prolonging the half-life of each protein. Knocking down E6^E7 expression in HPV16-positive CaSki cells by a splice junction-specific small interfering RNA (siRNA) destabilizes E6 and E7 and prevents cell growth. The same is true for the cells with a GRP78 knockdown or in the presence of an HSP90 inhibitor. Moreover, mapping and alignment analyses for splicing elements in 36 alpha-HPVs (α-HPVs) suggest the possible expression of E6^E7 mostly by other oncogenic or possibly oncogenic α-HPVs (HPV18, -30, -31, -39, -42, -45, -56, -59, -70, and -73). HPV18 E6^E7 is detectable in HPV18-positive HeLa cells and HPV18-infected raft tissues. All together, our data indicate that viral E6^E7 and cellular GRP78 or HSP90 might be novel targets for cervical cancer therapy.
IMPORTANCE
HPV16 is the most prevalent HPV genotype, being responsible for 60% of invasive cervical cancer cases worldwide. What makes HPV16 so potent in the development of cervical cancer remains a mystery. We discovered in this study that, besides producing two well-known oncoproteins, E6 and E7, seen in other high-risk HPVs, HPV16 produces E6^E7, a novel splice isoform of E6 and E7. E6^E7, in addition to self-interacting, binds cellular chaperone proteins, HSP90 and GRP78, and viral E6 and E7 to increase the steady-state levels and half-lives of viral oncoproteins, leading to cell proliferation. The splicing cis elements in the regulation of HPV16 E6^E7 production are highly conserved in 11 oncogenic or possibly oncogenic HPVs, and we confirmed the production of HPV18 E6^E7 in HPV18-infected cells. This study provides new insight into the mechanism of splicing, the interplay between different products of the polycistronic viral message, and the role of the host chaperones as they function.
doi:10.1128/mBio.02068-14
PMCID: PMC4337564  PMID: 25691589
4.  Human Papillomavirus Type 58 Genome Variations and RNA Expression in Cervical Lesions 
Journal of Virology  2013;87(16):9313-9322.
Human papillomavirus type 58 (HPV58) is relatively prevalent in China and other Asian countries. In this study, the HPV58 genome in cervical lesions was decoded from five grade 2 or 3 cervical intraepithelial neoplasia lesion (CIN2/3) samples and five cervical cancer tissues using rolling-circle amplification of total cell DNA and deep sequencing and verified by whole-genome cloning and sequencing. HPV58 isolates from China feature a total of 52 nucleotide substitutions (0.66%) from the reference HPV58 sequence, which appear mainly in two regions, with 12 from nucleotides (nt) 3430 to 4136 covering the E2/E4/E5 open reading frames (ORFs) and 13 from nt 4621 to 5540 covering the L2 ORF; these could be grouped as HPV58 Chinese Zhejiang-1, -2, and -3 (CNZJ-1, -2, and -3) according to their sequence similarities and restriction enzyme digestion. Phylogenetically, CNZJ-3 is similar to the reference HPV58 sublineage A1 sequence. The other two are close to sublineage A2. Analysis of cervical lesion-derived RNA revealed abundant HPV58 early transcripts spliced at the E6 and E1/E2 ORFs, where two 5′ splice sites at nt 232 and nt 898 and two 3′ splice sites at nt 510 and nt 3355 can be identified. Thus, our study represents the first genome-wide analysis of HPV58 and its expression in cervical lesions.
doi:10.1128/JVI.01154-13
PMCID: PMC3754072  PMID: 23785208
5.  Interplay between Polyadenylate-Binding Protein 1 and Kaposi's Sarcoma-Associated Herpesvirus ORF57 in Accumulation of Polyadenylated Nuclear RNA, a Viral Long Noncoding RNA 
Journal of Virology  2013;87(1):243-256.
Polyadenylate-binding protein cytoplasmic 1 (PABPC1) is a cytoplasmic-nuclear shuttling protein important for protein translation initiation and both RNA processing and stability. We report that PABPC1 forms a complex with the Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 protein, which allows ORF57 to interact with a 9-nucleotide (nt) core element of KSHV polyadenylated nuclear (PAN) RNA, a viral long noncoding RNA (lncRNA), and increase PAN stability. The N-terminal RNA recognition motifs (RRMs) of PABPC1 are necessary for the direct interaction with ORF57. During KSHV lytic infection, the expression of viral ORF57 leads to a substantial decrease in overall PABPC1 expression, along with a shift in the cellular distribution of the remaining PABPC1 to the nucleus. Interestingly, PABPC1 and ORF57 have opposing functions in modulating PAN steady-state accumulation. The suppressive effect of PABPC1 specific to PAN expression is alleviated by small interfering RNA knockdown of PABPC1 or by overexpression of ORF57. Conversely, ectopic PABPC1 reduces ORF57 steady-state protein levels and induces aberrant polyadenylation of PAN and thereby indirectly inhibits ORF57-mediated PAN accumulation. However, E1B-AP5 (heterogeneous nuclear ribonucleoprotein U-like 1), which interacts with a region outside the 9-nt core to stimulate PAN expression, does not interact or even colocalize with ORF57. Unlike PABPC1, the nuclear distribution of E1B-AP5 remains unchanged by viral lytic infection or overexpression of ORF57. Together, these data indicate that PABPC1 is an important cellular target of viral ORF57 to directly upregulate PAN accumulation during viral lytic infection, and the ability of host PABPC1 to disrupt ORF57 expression is a strategic host counterbalancing mechanism.
doi:10.1128/JVI.01693-12
PMCID: PMC3536381  PMID: 23077296
6.  Kaposi sarcoma-associated herpesviral IL6 and human IL6 open reading frames contain miRNA binding sites and are subject to cellular miRNA regulation 
The Journal of pathology  2011;225(3):378-389.
Kaposi sarcoma-associated herpesvirus (KSHV) encodes a viral interleukin 6 (vIL6) that mimics many activities of human IL6 (hIL6). Both vIL6 and hIL6 play important roles in stimulating the proliferation of tumors caused by KSHV. Here, we provide evidence that a miRNA pathway is involved in regulation of vIL6 and hIL6 expression through binding sites in their open reading frames (ORF). We show a direct repression of vIL6 by hsa-miR-1293 and hIL6 by hsa-miR-608. The repression of vIL6 by miR-1293 was reversed by disruption of the vIL6 miR-1293 seed match through the introduction of point mutations. In addition, expression of vIL6 or hIL6 in KSHV-infected cells could be enhanced by transfection of the respective miRNA inhibitors. In situ hybridization of human lymph node sections revealed that miR-1293 is primarily expressed in the germinal center, but is deficient in the mantle zone of lymph nodes where the expression of vIL6 is often found in patients with KSHV-associated multicentric Castleman’s disease, providing evidence of an anatomic correlation. Together, our study indicates that IL6 expression can be regulated by miRNA interactions in its ORF and provides evidence for the role of these interactions in the pathogenesis of KSHV-associated diseases.
doi:10.1002/path.2962
PMCID: PMC3528401  PMID: 21984125
Viral IL6; IL6; miRNA; Kaposi sarcoma-associated herpesvirus; germinal center; gene expression; post-transcriptional regulation
7.  Up-Regulation of p18Ink4c Expression by Oncogenic HPV E6 via p53-miR-34a Pathway 
Binding of p53 to miR-34a promoter activates the expression of tumor suppressive miR-34a. Oncogenic HPV infection downregulates miR-34a expression through viral E6 degradation of p53. In this report, we found that miR-34a specifically targets p18Ink4c, a CDK4 and CDK6 inhibitor induced by E2F transactivation. HPV18+ HeLa cells with ectopic miR-34a expression or by E6 siRNA knockdown-induced expression of endogenous miR-34a exhibited a substantial reduction of p18Ink4c in a dose-dependent manner, but had no effect on p16Ink4a, another member of CDK4/6 inhibitor family. In contrast, de novo infection by oncogenic HPVs of human keratinocyte-derived raft tissues increased p18Ink4c expression. Suppression of endogenous miR-34a in cell lines with a miR-34a inhibitor also increased p18Ink4c. We found that miR-34a suppresses the expression of p18Ink4c by binding to a specific seed match in the 5' UTR of p18Ink4c. Further investigation found remarkable increase of p18Ink4c in cervical precancer lesions and cervical cancer. Immunohistochemical staining of cervical tissue arrays showed increased expression of p18Ink4c in 68% of cervical cancer, 8.3% of chronic cervical inflammation, and 4.8% of normal cervix. Although p18Ink4c inhibits cell proliferation in general and regulates E2F1 expression in HCT116 cells, it appears not to function as a tumor suppressor in cervical cancer cells lacking an intact G1 checkpoint due to viral E7 degradation of pRB. In summary, this study demonstrates an intimate connection among oncogenic HPV E6, p53, miR-34a, and p18Ink4c and identifies p18Ink4c as a possible biomarker for cervical cancer.
doi:10.1002/ijc.25800
PMCID: PMC3086996  PMID: 21128241
Human papillomaviruses; microRNAs; p53; cell cycle control; p18ink4c
8.  Requirement of UAP56, URH49, RBM15, and OTT3 in expression of Kaposi sarcoma-associated herpesvirus ORF57 
Virology  2010;407(2):206-212.
Transport of mRNA from the nucleus to the cytoplasm is mediated by cellular RNA export factors. In this report, we examined how RNA export factors UAP56 and URH49, and RNA export cofactors RBM15 and OTT3, function in modulating KSHV ORF57 expression. We found that knockdown of each factor by RNAi led to decreased ORF57 expression. Specifically, reduced expression of either UAP56 or RBM15 led to nuclear export deficiency of ORF57 RNA. In the context of the KSHV genome, the near absence of UAP56 or RBM15 reduced the expression of both ORF57 and ORF59 (an RNA target of ORF57), but not ORF50. Collectively, our data indicate that the expression of KSHV ORF57 is regulated by cellular RNA export factors and cofactors at the posttranscriptional level.
doi:10.1016/j.virol.2010.08.014
PMCID: PMC2952739  PMID: 20828777
9.  SRp20 is a proto-oncogene critical for cell proliferation and tumor induction and maintenance 
Tumor cells display a different profile of gene expression than their normal counterparts. Perturbations in the levels of cellular splicing factors can alter gene expression, potentially leading to tumorigenesis. We found that splicing factor SRp20 (SFRS3) is highly expressed in cancers. SRp20 regulated the expression of Forkhead box transcription factor M1 (FoxM1) and two of its transcriptional targets, PLK1 and Cdc25B, and controlled cell cycle progression and proliferation. Cancer cells with RNAi-mediated reduction of SRp20 expression exhibited G2/M arrest, growth retardation, and apoptosis. Increased SRp20 expression in rodent fibroblasts promoted immortal cell growth and transformation. More importantly, we found that SRp20 promoted tumor induction and the maintenance of tumor growth in nude mice and rendered immortal rodent fibroblasts tumorigenic. Collectively, these results suggest that increased SRp20 expression in tumor cells is a critical step for tumor initiation, progression, and maintenance.
PMCID: PMC3005347  PMID: 21179588
Cancer; splicing factors; SFRS3; SRp20; G2/M arrest; cell transformation; tumor induction
10.  Regulation of bovine papillomavirus type 1 gene expression by RNA processing 
Bovine papillomavirus type 1 (BPV-1) has served as a prototype for studying the molecular biology and pathogenesis of papillomaviruses. The expression of BPV-1 early and late genes is highly regulated at both transcription and post-transcriptional levels and strictly tied to the differentiation of keratinocytes. BPV-1 infects keratinocytes in the basal layer of the skin and replicates in the nucleus of infected cells in a differentiation-dependent manner. Although viral early genes begin to be expressed from the infected, undifferentiated basal cells, viral late genes are not expressed until the infected cells enter the terminal differentiation stage. Both BPV-1 early and late transcripts are intron-containing bicistronic or polycistronic RNAs, bearing more than one open reading frame and are polyadenylated at either an early or late poly (A) site. Nuclear RNA processing of these transcripts by RNA splicing and poly (A) site selection has been extensively analyzed in the past decade and various viral cis-elements and cellular factors involved in regulation of viral RNA processing were discovered, leading to our better understanding of the gene expression and biology of human papillomaviruses.
PMCID: PMC2654602  PMID: 19273129
Papillomaviruses; Gene expression; RNA splicing; RNA polyadenylation; Post-transcriptional regulation; Review
11.  Kaposi’s sarcoma-associated herpesvirus ORF57 in viral RNA processing 
Kaposi’s sarcoma-associated herpesvirus (KSHV) ORF57 (MTA, mRNA transcript accumulation) is a multifunctional regulator of the expression of viral lytic genes. KSHV ORF57 is expressed during viral lytic infection and is essential for virus production. Like its homologues in the herpesvirus family, ORF57 promotes the accumulation (stabilization) and export of viral intronless RNA transcripts by a mechanism which remains to be defined. The ORF57-Aly/REF interaction plays only a small role in viral RNA export. Although other members of the family generally inhibit the splicing of cellular RNAs, KSHV ORF57 and EBV EB2, in sharp contrast, stimulate viral RNA splicing for the expression of viral intron-containing genes. The functions of KSHV ORF57 are independent of transcription and of other viral proteins; instead, these functions always rely on cellular components and occur in various protein-RNA complexes. ORF57 may synergize with KSHV ORF50 to transactivate a subset of viral promoters by an unknown mechanism. Thus, some functions of ORF57 have been conserved while others have diverged from its homologues as ORF57 adapted over evolution to KSHV biology and pathogenesis.
PMCID: PMC2654597  PMID: 19273144
Kaposi’s sarcoma-associated herpesvirus; Gene expression; ORF57; RNA splicing; Post-transcriptional regulation; Protein-RNA interaction; RNA export; Review
12.  Regulation of alternative RNA splicing by exon definition and exon sequences in viral and mammalian gene expression 
Journal of biomedical science  2004;11(3):278-294.
Intron removal from a pre-mRNA by RNA splicing was once thought to be controlled mainly by intron splicing signals. However, viral and other eukaryotic RNA exon sequences have recently been found to regulate RNA splicing, polyadenylation, export, and nonsense-mediated RNA decay in addition to their coding function. Regulation of alternative RNA splicing by exon sequences is largely attributable to the presence of two major cis-acting elements in the regulated exons, the exonic splicing enhancer (ESE) and the suppressor or silencer (ESS). Two types of ESEs have been verified from more than 50 genes or exons: purine-rich ESEs, which are the more common, and non-purine-rich ESEs. In contrast, the sequences of ESSs identified in approximately 21 genes or exons are highly diverse and show little similarity to each other. Through interactions with cellular splicing factors, an ESE or ESS determines whether or not a regulated splice site, usually an upstream 3′ splice site, will be used for RNA splicing. However, how these elements function precisely in selecting a regulated splice site is only partially understood. The balance between positive and negative regulation of splice site selection likely depends on the cis-element’s identity and changes in cellular splicing factors under physiological or pathological conditions.
doi:10.1159/000077096
PMCID: PMC2442652  PMID: 15067211
RNA; exons; introns; alternative RNA splicing; gene expression; RNA processing; splicing enhancers; splicing suppressors
13.  Short-term induction and long-term suppression of HPV16 oncogene silencing by RNA interference in cervical cancer cells 
Oncogene  2006;25(14):2094-2104.
RNA interference-mediated gene silencing has the potential to block gene expression. A synthetic double-stranded (ds) siRNA based on a sequence motif of 21 nts from human papillomavirus 16 (HPV16) E6E7 bicistronic RNA was found to be a potent small interfering RNA (siRNA) that suppresses expression of both the E6 and E7 oncogenes in HPV16+ CaSki and SiHa cells. When stably expressed as a short hairpin RNA (shRNA) in these cells, however, siRNA silencing of E6 and E7 expression was efficient only at early cell passages, but became inefficient with increased cell passages despite the continued expression of the siRNA at the same level. The loss of the siRNA function was duplicable in stable p53 siRNA cells, but not in stable lamin A/C siRNA cells, suggesting that it is gene-selective. The cells resistant to siRNA function retained normal siRNA processing, duplex unwinding and degradation of the unwound sense strand and RISC formation, suggesting that loss of the siRNA function occurred at a later step. Surprisingly, the siRNA-resistant cells were found to express notably a cytoplasmic protein of ~50 kDa that specifically and characteristically interacted with the unwound, antisense strand E7 siRNA. Altogether, our data indicate that a potent siRNA targeting to an essential or regulatory gene might induce a cell to develop siRNA-suppressive function.
doi:10.1038/sj.onc.1209244
PMCID: PMC1800826  PMID: 16369495
HPV16; E6; E7; oncogene; siRNA; cervical cancer
14.  Colorectal papillomavirus infection in colorectal cancer patients 
Purpose: Infection with human papillomaviruses (HPVs) is associated with the development of cervical cancer, but whether HPVs have a role in colorectal cancer remains controversial.
Experimental Design: To determine the relationship between HPV and colorectal cancer, we performed a retrospective, controlled study using tumor and tumor-adjacent colorectal tissues dissected from patients with colorectal cancer, as well as colorectal tissues from control individuals with no cancer. The samples were processed in a blinded fashion for nested PCR and in situ PCR detection of HPV DNAs. The PCR products were gel purified and sequenced for HPV genotyping.
Results: We found that colorectal tissues from 28 (51%) of 55 patients with colorectal cancer were positive for HPV DNA. Colorectal tissues from all 10 control individuals were negative for HPV DNA (P=0.0034). Of the 107 usable (GAPDH+) samples collected as paired colorectal tissues (tumor and tumor-adjacent tissues) from the patients, 38 (36%) had HPV16 (n=31), HPV18 (n=5), or HPV45 (n=2), with HPV DNA in both tumor and tumor-adjacent tissues of 10 paired samples, 13 in only the tumor, and 5 in only tumor-adjacent tissues. In situ PCR detection of the tumor tissues confirmed the presence of HPV DNA in tumor cells.
Conclusion: Our results suggest that colorectal HPV infection is common in patients with colorectal cancer, albeit at a low DNA copy number, with HPV16 being the most prevalent type. HPV infection may play a role in colorectal carcinogenesis.
doi:10.1158/1078-0432.CCR-04-1680
PMCID: PMC1479314  PMID: 15837733
Human papillomavirus type 16; Viral oncogenesis; Tumor virus infection; PCR
15.  PAPILLOMAVIRUS GENOME STRUCTURE, EXPRESSION, AND POST-TRANSCRIPTIONAL REGULATION 
Papillomaviruses are a group of small non-enveloped DNA tumor viruses whose infection usually causes benign epithelial lesions (warts). Certain types of HPVs, such as HPV-16, HPV-18, and HPV-31, have been recognized as causative agents of cervical cancer and anal cancer and their infections, which arise via sexual transmission, are associated with more than 95% of cervical cancer. Papillomaviruses infect keratinocytes in the basal layer of stratified squamous epithelia and replicate in the nucleus of infected keratinocytes in a differentiation-dependent manner. Viral gene expression in infected cells depends on cell differentiation and is tightly regulated at the transcriptional and post-transcriptional levels. A noteworthy feature of all papillomavirus transcripts is that they are transcribed as a bicistronic or polycistronic form containing two or more ORFs and are polyadenylated at either an early or late poly(A) site. In the past ten years, remarkable progress has been made in understanding how this complex viral gene expression is regulated at the level of transcription (such as via DNA methylation) and particularly post-transcription (including RNA splicing, polyadenylation, and translation). Current knowledge of papillomavirus mRNA structure and RNA processing has provided some clues on how to control viral oncogene expression. However, we still have little knowledge about which mRNAs are used to translate each viral protein. Continuing research on post-transcriptional regulation of papillomavirus infection will remain as a future focus to provide more insights into papillomavirus-host interactions, the virus life-cycle, and viral oncogenesis.
PMCID: PMC1472295  PMID: 16720315
papillomaviruses; gene expression; RNA splicing; RNA polyadenylation; posttranscriptional regulation
16.  Utilization of the Bovine Papillomavirus Type 1 Late-Stage-Specific Nucleotide 3605 3′ Splice Site Is Modulated by a Novel Exonic Bipartite Regulator but Not by an Intronic Purine-Rich Element 
Journal of Virology  2000;74(22):10612-10622.
Bovine papillomavirus type 1 (BPV-1) late gene expression is regulated at both transcriptional and posttranscriptional levels. Maturation of the capsid protein (L1) pre-mRNA requires a switch in 3′ splice site utilization. This switch involves activation of the nucleotide (nt) 3605 3′ splice site, which is utilized only in fully differentiated keratinocytes during late stages of the virus life cycle. Our previous studies of the mechanisms that regulate BPV-1 alternative splicing identified three cis-acting elements between these two splice sites. Two purine-rich exonic splicing enhancers, SE1 and SE2, are essential for preferential utilization of the nt 3225 3′ splice site at early stages of the virus life cycle. Another cis-acting element, exonic splicing suppressor 1 (ESS1), represses use of the nt 3225 3′ splice site. In the present study, we investigated the late-stage-specific nt 3605 3′ splice site and showed that it has suboptimal features characterized by a nonconsensus branch point sequence and a weak polypyrimidine track with interspersed purines. In vitro and in vivo experiments showed that utilization of the nt 3605 3′ splice site was not affected by SE2, which is intronically located with respect to the nt 3605 3′ splice site. The intronic location and sequence composition of SE2 are similar to those of the adenovirus IIIa repressor element, which has been shown to inhibit use of a downstream 3′ splice site. Further studies demonstrated that the nt 3605 3′ splice site is controlled by a novel exonic bipartite element consisting of an AC-rich exonic splicing enhancer (SE4) and an exonic splicing suppressor (ESS2) with a UGGU motif. Functionally, this newly identified bipartite element resembles the bipartite element composed of SE1 and ESS1. SE4 also functions on a heterologous 3′ splice site. In contrast, ESS2 functions as an exonic splicing suppressor only in a 3′-splice-site-specific and enhancer-specific manner. Our data indicate that BPV-1 splicing regulation is very complex and is likely to be controlled by multiple splicing factors during keratinocyte differentiation.
PMCID: PMC110935  PMID: 11044105
17.  A genome landscape of SRSF3-regulated splicing events and gene expression in human osteosarcoma U2OS cells 
Nucleic Acids Research  2015;44(4):1854-1870.
Alternative RNA splicing is an essential process to yield proteomic diversity in eukaryotic cells, and aberrant splicing is often associated with numerous human diseases and cancers. We recently described serine/arginine-rich splicing factor 3 (SRSF3 or SRp20) being a proto-oncogene. However, the SRSF3-regulated splicing events responsible for its oncogenic activities remain largely unknown. By global profiling of the SRSF3-regulated splicing events in human osteosarcoma U2OS cells, we found that SRSF3 regulates the expression of 60 genes including ERRFI1, ANXA1 and TGFB2, and 182 splicing events in 164 genes, including EP300, PUS3, CLINT1, PKP4, KIF23, CHK1, SMC2, CKLF, MAP4, MBNL1, MELK, DDX5, PABPC1, MAP4K4, Sp1 and SRSF1, which are primarily associated with cell proliferation or cell cycle. Two SRSF3-binding motifs, CCAGC(G)C and A(G)CAGCA, are enriched to the alternative exons. An SRSF3-binding site in the EP300 exon 14 is essential for exon 14 inclusion. We found that the expression of SRSF1 and SRSF3 are mutually dependent and coexpressed in normal and tumor tissues/cells. SRSF3 also significantly regulates the expression of at least 20 miRNAs, including a subset of oncogenic or tumor suppressive miRNAs. These data indicate that SRSF3 affects a global change of gene expression to maintain cell homeostasis.
doi:10.1093/nar/gkv1500
PMCID: PMC4770227  PMID: 26704980
18.  Vemurafenib-resistant BRAF selects alternative branch points different from its wild-type BRAF in intron 8 for RNA splicing 
Cell & Bioscience  2015;5:70.
One mechanism of resistance of the melanoma-associated BRAF kinase to its small molecule inhibitor vemurafenib is by point mutations in its intron 8 resulting in exons 4–8 skipping. In this report, we carried out in vitro BRAF RNA splicing assays and lariat RT-PCR to map the intron 8 branch points in wild-type and BRAF mutants. We identify multiple branch points (BP) in intron 8 of both wild-type (wt) and vemurafenib-resistant BRAF RNA. In wt BRAF, BPs are located at -29A, -28A and -26A, whereas in a vemurafenib-resistant BRAF splicing mutant, BPs map to -22A, -18A and -15A, proximal to the intron 8 3′ splice site. This finding of a distal-to-proximal shift of the branch point sequence in BRAF splicing in response to point-mutations in intron 8 provides insight into the regulation of BRAF alternative splicing upon vemurafenib resistance.
Electronic supplementary material
The online version of this article (doi:10.1186/s13578-015-0061-7) contains supplementary material, which is available to authorized users.
doi:10.1186/s13578-015-0061-7
PMCID: PMC4687071  PMID: 26697165
BRAF; RNA splicing; Branch point mapping; Lariat RT-PCR; Vemurafenib-resistance
19.  Split genes and their expression in Kaposi’s sarcoma-associated herpesvirus 
Reviews in medical virology  2003;13(3):173-184.
SUMMARY
A split or interrupted gene is defined as a gene consisting of introns and exons. Removal (splicing) of the intron (s) from a primary transcript (pre-mRNA) is an essential process to create a mRNA. Initial assignment of a potential protein coding region in KSHV genome was based on initiation codon context and predicted protein size larger than 100 amino acids, but the gene discontinuity was disregarded. Experimental investigation of the assigned ORFs has demonstrated that there are up to 25 split genes, more than one fourth of the total KSHV genes described in KSHV genome. This includes the genes involved in all phases (latent, immediate early, early and late) of KSHV infection. The complexity of a split gene expression depends upon the availability of a proximal promoter and polyadenylation (pA) signal. Sharing a single promoter or a single pA signal by two or three genes is not uncommon in the expression of KSHV split genes and the resulting transcripts are usually polycistronic. Among those of KSHV split genes, 15 genes express a bicistronic or tricistronic RNA and 10 genes express a monocistronic RNA. Alternative RNA splicing could happen in a particular pre-mRNA due to intron or exon inclusion or skipping or the presence of an alternative 5′ ss or 3′ ss. This may, respectively, result in at least 8 species of K8 and 14 species of K15 transcripts. This appears to be related to cell differentiation and stages of the virus infection, presumably involving in viral cis elements and trans splicing factors.
doi:10.1002/rmv.387
PMCID: PMC4681429  PMID: 12740832
20.  Both Decreased and Increased SRPK1 Levels Promote Cancer by Interfering with PHLPP-Mediated Dephosphorylation of Akt 
Molecular cell  2014;54(3):378-391.
Akt activation is a hallmark of human cancers. Here we report a critical mechanism for regulation of Akt activity by the splicing kinase SRPK1, a downstream Akt target for transducing growth signals to regulate splicing. Surprisingly, we find that SRPK1 has a tumor suppressor function because ablation of SRPK1 in mouse embryonic fibroblasts induces cell transformation. We link the phenotype to constitutive Akt activation from genome-wide phosphoproteomics analysis and discover that down-regulated SRPK1 impairs the recruitment of the Akt phosphatase PHLPP1 to Akt. Interestingly, SRPK1 overexpression is also tumorigenic because excess SRPK1 squelches PHLPP1. Thus, aberrant SRPK1 expression in either direction induces constitutive Akt activation, providing a mechanistic basis for previous observations that SRPK1 is down-regulated in some cancer contexts and up-regulated in others.
doi:10.1016/j.molcel.2014.03.007
PMCID: PMC4019712  PMID: 24703948
SR protein-specific kinases; oncogene; tumor suppressor; Akt activation; Akt phosphatase PHLPP1
21.  Multiple Regions of Kaposi’s Sarcoma-Associated Herpesvirus ORF59 RNA are Required for Its Expression Mediated by Viral ORF57 and Cellular RBM15 
Viruses  2015;7(2):496-510.
KSHV ORF57 (MTA) promotes RNA stability of ORF59, a viral DNA polymerase processivity factor. Here, we show that the integrity of both ORF59 RNA ends is necessary for ORF57-mediated ORF59 expression and deletion of both 5’ and 3’ regions, or one end region with a central region, of ORF59 RNA prevents ORF57-mediated translation of ORF59. The ORF59 sequence between nt 96633 and 96559 resembles other known MTA-responsive elements (MREs). ORF57 specifically binds to a stem-loop region from nt 96596–96572 of the MRE, which also binds cellular RBM15. Internal deletion of the MRE from ORF59 led to poor export, but accumulation of nuclear ORF59 RNA in the presence of ORF57 or RBM15. Despite of being translatable in the presence of ORF57, this deletion mutant exhibits translational defect in the presence of RBM15. Together, our results provide novel insight into the roles of ORF57 and RBM15 in ORF59 RNA accumulation and protein translation.
doi:10.3390/v7020496
PMCID: PMC4353900  PMID: 25690794
KSHV; ORF59; ORF57; RNA accumulation; RNA export; RBM15
22.  KSHV ORF57, a Protein of Many Faces 
Viruses  2015;7(2):604-633.
Kaposi’s sarcoma-associated herpesvirus (KSHV) ORF57 protein (also known as mRNA transcript accumulation (Mta)) is a potent posttranscriptional regulator essential for the efficient expression of KSHV lytic genes and productive KSHV replication. ORF57 possesses numerous activities that promote the expression of viral genes, including the three major functions of enhancement of RNA stability, promotion of RNA splicing, and stimulation of protein translation. The multifunctional nature of ORF57 is driven by its ability to interact with an array of cellular cofactors. These interactions are required for the formation of ORF57-containing ribonucleoprotein complexes at specific binding sites in the target transcripts, referred as Mta-responsive elements (MREs). Understanding of the ORF57 protein conformation has led to the identification of two structurally-distinct domains within the ORF57 polypeptide: an unstructured intrinsically disordered N-terminal domain and a structured α-helix-rich C-terminal domain. The distinct structures of the domains serve as the foundation for their unique binding affinities: the N-terminal domain mediates ORF57 interactions with cellular cofactors and target RNAs, and the C-terminal domain mediates ORF57 homodimerization. In addition, each domain has been found to contribute to the stability of ORF57 protein in infected cells by counteracting caspase- and proteasome-mediated degradation pathways. Together, these new findings provide insight into the function and biological properties of ORF57 in the KSHV life cycle and pathogenesis.
doi:10.3390/v7020604
PMCID: PMC4353906  PMID: 25674768
KSHV; ORF57 protein; viral expression; posttranscriptional regulator; RNA processing
23.  Adapted Resistance to the Knockdown Effect of shRNA-Derived Srsf3 siRNAs in Mouse Littermates 
Gene silencing techniques are widely used to control gene expression and have potential for RNAi-based therapeutics. In this report, transgenic mouse lines were created for conditional knockdown of Srsf3 (SRp20) expression in liver and mammary gland tissues by expressing Srsf3-specific shRNAs driven by a U6 promoter. Although a small portion of the transgenic mouse littermates were found to produce siRNAs in the targeted tissues, most of the transgenic littermates at two months of age failed to display a knockdown phenotype of Srsf3 expression in their liver and mammary gland tissues where an abundant level of Srsf3 siRNAs remained. We saw only one of four mice with liver/mammary gland expressing Srsf3 siRNA displayed a suppressed level of Srsf3 protein, but not the mRNA. Data indicate that the host resistance to a gene-specific siRNA targeting an essential gene transcript can be developed in animals, presumably as a physiological necessity to cope with the hostile perturbation.
doi:10.7150/ijbs.13011
PMCID: PMC4582148  PMID: 26435690
Srsf3; shRNAs; transgenic mouse
24.  Oncogenes and RNA splicing of human tumor viruses 
Approximately 10.8% of human cancers are associated with infection by an oncogenic virus. These viruses include human papillomavirus (HPV), Epstein–Barr virus (EBV), Merkel cell polyomavirus (MCV), human T-cell leukemia virus 1 (HTLV-1), Kaposi's sarcoma-associated herpesvirus (KSHV), hepatitis C virus (HCV) and hepatitis B virus (HBV). These oncogenic viruses, with the exception of HCV, require the host RNA splicing machinery in order to exercise their oncogenic activities, a strategy that allows the viruses to efficiently export and stabilize viral RNA and to produce spliced RNA isoforms from a bicistronic or polycistronic RNA transcript for efficient protein translation. Infection with a tumor virus affects the expression of host genes, including host RNA splicing factors, which play a key role in regulating viral RNA splicing of oncogene transcripts. A current prospective focus is to explore how alternative RNA splicing and the expression of viral oncogenes take place in a cell- or tissue-specific manner in virus-induced human carcinogenesis.
doi:10.1038/emi.2014.62
PMCID: PMC4185361  PMID: 26038756
EBV; HBV; HCV; HPV; HTLV-1; KSHV; MCV; RNA splicing
25.  Genome sequencing accuracy by RCA-seq versus long PCR template cloning and sequencing in identification of human papillomavirus type 58 
Cell & Bioscience  2014;4:5.
Background
Genome variations in human papillomaviruses (HPVs) are common and have been widely investigated in the past two decades. HPV genotyping depends on the finding of the viral genome variations in the L1 ORF. Other parts of the viral genome variations have also been implicated as a possible genetic factor in viral pathogenesis and/or oncogenicity.
Results
In this study, the HPV58 genome in cervical lesions was completely sequenced both by rolling-circle amplification of total cell DNA and deep sequencing (RCA-seq) and by long PCR template cloning and sequencing. By comparison of three HPV58 genome sequences decoded from three clinical samples to reference HPV-58, we demonstrated that RCA-seq is much more accurate than long-PCR template cloning and sequencing in decoding HPV58 genome. Three HPV58 genomes decoded by RCA-seq displayed a total of 52 nucleotide substitutions from reference HPV58, which could be verified by long PCR template cloning and sequencing. However, the long PCR template cloning and sequencing led to additional nucleotide substitutions, insertions, and deletions from an authentic HPV58 genome in a clinical sample, which vary from one cloned sequence to another. Because the inherited error-prone nature of Tgo DNA polymerase used in preparation of the long PCR templates of HPV58 genome from the clinical samples, the measurable error rate in incorporation of nucleotide into an elongating DNA template was about 0.149% ±0.038% in our studies.
Conclusions
Since PCR template cloning and sequencing is widely used in identification of single nucleotide polymorphism (SNP), our data indicate that a serious caution should be taken in finding of true SNPs in various genetic studies.
doi:10.1186/2045-3701-4-5
PMCID: PMC3903022  PMID: 24410913
Human papillomaviruses; HPV58; Cervical cancer; Single nucleotide polymorphism; Genotyping; Genome variations; Rolling circle amplification; DNA deep sequencing

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