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.
Viral IL6; IL6; miRNA; Kaposi sarcoma-associated herpesvirus; germinal center; gene expression; post-transcriptional regulation
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.
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.
Papillomaviruses; Gene expression; RNA splicing; RNA polyadenylation; Post-transcriptional regulation; Review
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.
Kaposi’s sarcoma-associated herpesvirus; Gene expression; ORF57; RNA splicing; Post-transcriptional regulation; Protein-RNA interaction; RNA export; Review
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.
RNA; exons; introns; alternative RNA splicing; gene expression; RNA processing; splicing enhancers; splicing suppressors
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.
HPV16; E6; E7; oncogene; siRNA; cervical cancer
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.
Human papillomavirus type 16; Viral oncogenesis; Tumor virus infection; PCR
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.
papillomaviruses; gene expression; RNA splicing; RNA polyadenylation; posttranscriptional regulation
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.
High-risk HPV infection leads to aberrant expression of cellular oncogenic and tumor suppressive miRNAs. A large number of these miRNA genes are downstream targets of the transcription factors c-Myc, p53, and E2F and their expression can therefore be modulated by oncogenic HPV E6 and E7. Cervical cancer represents an unique tumor model for understanding how viral E6 and E7 oncoproteins deregulate the expression of the miR-15/16 cluster, miR-17-92 family, miR-21, miR-23b, miR-34a, and miR-106b/93/25 cluster via the E6–p53 and E7–pRb pathways. Moreover, miRNAs may influence the expression of papillomavirus genes in a differentiation-dependent manner by targeting viral RNA transcripts. Cellular miRNAs affecting HPV DNA replication are of great interest and will be a future focus. We are entering an era focusing on miRNA and noncoding RNA, and the study of HPV and host miRNA interactions will continue to shed more light on our understanding of the HPV life cycle and the mechanistic underpinnings of HPV-induced oncogenesis.
human papillomaviruses; microRNAs; oncogenes; tumor suppressor genes; cervical cancer; gene expression
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.
Human papillomaviruses; microRNAs; p53; cell cycle control; p18ink4c
Erythromelagia is a condition characterized by attacks of burning pain and inflammation in the extremeties. An epidemic form of this syndrome occurs in secondary students in rural China and a virus referred to as erythromelalgia-associated poxvirus (ERPV) was reported to have been recovered from throat swabs in 1987. Studies performed at the time suggested that ERPV belongs to the orthopoxvirus genus and has similarities with ectromelia virus, the causative agent of mousepox. We have determined the complete genome sequence of ERPV and demonstrated that it has 99.8% identity to the Naval strain of ectromelia virus and a slighly lower identity to the Moscow strain. Small DNA deletions in the Naval genome that are absent from ERPV may suggest that the sequenced strain of Naval was not the immediate progenitor of ERPV.
Human papillomavirus type 18 (HPV18) is the second most common oncogenic HPV genotype, responsible for ∼15% of cervical cancers worldwide. In this study, we constructed a full HPV18 transcription map using HPV18-infected raft tissues derived from primary human vaginal or foreskin keratinocytes. By using 5′ rapid amplification of cDNA ends (RACE), we mapped two HPV18 transcription start sites (TSS) for early transcripts at nucleotide (nt) 55 and nt 102 and the HPV18 late TSS frequently at nt 811, 765, or 829 within the E7 open reading frame (ORF) of the virus genome. HPV18 polyadenylation cleavage sites for early and late transcripts were mapped to nt 4270 and mainly to nt 7299 or 7307, respectively, by using 3′ RACE. Although all early transcripts were cleaved exclusively at a single cleavage site, HPV18 late transcripts displayed the heterogeneity of 3′ ends, with multiple minor cleavage sites for late RNA polyadenylation. HPV18 splice sites/splice junctions for both early and late transcripts were identified by 5′ RACE and primer walking techniques. Five 5′ splice sites (donor sites) and six 3′ splice sites (acceptor sites) that are highly conserved in other papillomaviruses were identified in the HPV18 genome. HPV18 L1 mRNA translates a L1 protein of 507 amino acids (aa), smaller than the 568 aa residues previously predicted. Collectively, a full HPV18 transcription map constructed from this report will lead us to further understand HPV18 gene expression and virus oncogenesis.
Kaposi's sarcoma-associated herpesvirus (KSHV) lytic infection increases the expression of viral and human interleukin-6 (vIL-6 and hIL-6, respectively), an important factor for cell growth and pathogenesis. Here, we report genome-wide analysis of viral RNA targets of KSHV ORF57 by a novel UV-cross-linking and immunoprecipitation (CLIP) assay. We identified 11 viral transcripts as putative ORF57 targets and demonstrate that vIL-6 mRNA is an authentic target of ORF57. Disrupting the ORF57 gene in the KSHV genome leads to inefficient expression of vIL-6. With transient transfection, the expression of vIL-6 could be enhanced greatly in the presence of ORF57 in a dose-dependent manner. We found that the open reading frame (ORF) region of vIL-6 RNA contains an MRE (MTA [ORF57]-responsive element) composed of two motifs, MRE-A and MRE-B, and binding of ORF57 to these two motifs stabilizes vIL-6 RNA and promotes vIL-6 translation. We demonstrate that vIL-6 MRE-B bears an miR-1293 binding site and that, mechanistically, ORF57 competes with miR-1293 for the same binding site to interact with vIL-6 RNA, thereby preventing vIL-6 RNA from association with the miR-1293-specified RNA-induced silencing complex (RISC). Consistent with this, ORF57 also interacts with an miR-608 binding site in the hIL-6 ORF and prevents miR-608 repression of hIL-6. Collectively, our results identify a novel function of ORF57 in being responsible for stabilization of viral and human IL-6 RNAs and the corresponding enhancement of RNA translation. In addition, our data provide the first evidence that a tumor virus may use a viral protein to interfere with microRNA (miRNA)-mediated repression of an miRNA target to induce cell proliferation and tumorigenesis during virus infection.
Kaposi's sarcoma-associated herpesvirus (KSHV) encodes ORF57, which promotes the accumulation of specific KSHV mRNA targets, including ORF59 mRNA. We report that the cellular export NXF1 cofactors RBM15 and OTT3 participate in ORF57-enhanced expression of KSHV ORF59. We also found that ectopic expression of RBM15 or OTT3 augments ORF59 production in the absence of ORF57. While RBM15 promotes the accumulation of ORF59 RNA predominantly in the nucleus compared to the levels in the cytoplasm, we found that ORF57 shifted the nucleocytoplasmic balance by increasing ORF59 RNA accumulation in the cytoplasm more than in the nucleus. By promoting the accumulation of cytoplasmic ORF59 RNA, ORF57 offsets the nuclear RNA accumulation mediated by RBM15 by preventing nuclear ORF59 RNA from hyperpolyadenylation. ORF57 interacts directly with the RBM15 C-terminal portion containing the SPOC domain to reduce RBM15 binding to ORF59 RNA. Although ORF57 homologs Epstein-Barr virus (EBV) EB2, herpes simplex virus (HSV) ICP27, varicella-zoster virus (VZV) IE4/ORF4, and cytomegalovirus (CMV) UL69 also interact with RBM15 and OTT3, EBV EB2, which also promotes ORF59 expression, does not function like KSHV ORF57 to efficiently prevent RBM15-mediated nuclear accumulation of ORF59 RNA and RBM15's association with polyadenylated RNAs. Collectively, our data provide novel insight elucidating a molecular mechanism by which ORF57 promotes the expression of viral intronless genes.
Mutations in two branch-point sequences (BPS) in intron 3 of the XPC DNA repair gene affect pre-mRNA splicing in association with xeroderma pigmentosum (XP) with many skin cancers (XP101TMA) or no skin cancer (XP72TMA), respectively. To investigate the mechanism of these abnormalities we now report that transfection of minigenes with these mutations revealed abnormal XPC pre-mRNA splicing that mimicked pre-mRNA splicing in the patients’ cells. DNA oligonucleotide-directed RNase H digestion demonstrated that mutations in these BPS disrupt U2 snRNP – BPS interaction. XP101TMA cells had no detectable XPC protein but XP72TMA had 29% of normal levels. A small amount of XPC protein was detected at sites of localized UV-damaged DNA in XP72TMA cells which then recruited other nucleotide excision repair (NER) proteins. In contrast, XP101TMA cells had no detectable recruitment of XPC or other NER proteins. Post-UV survival and photoproduct assays revealed greater reduction in DNA repair in XP101TMA cells than in XP72TMA. Thus mutations in XPC BPS resulted in disruption of U2 snRNP-BPS interaction leading to abnormal pre-mRNA splicing and reduced XPC protein. At the cellular level these changes were associated with features of reduced DNA repair including diminished NER protein recruitment, reduced post-UV survival and impaired photoproduct removal.
XPC; DNA repair; pre-mRNA splicing; xeroderma pigmentosum; skin cancer; U2 snRNP
Kaposi sarcoma-associated herpesvirus (KSHV) ORF57, also known as Mta (mRNA transcript accumulation), enhances viral intron-less transcript accumulation and promotes splicing of intron-containing viral RNA transcripts. In this study, we identified KSHV PAN, a long non-coding polyadenylated nuclear RNA as a main target of ORF57 by a genome-wide CLIP (cross-linking and immunoprecipitation) approach. KSHV genome lacking ORF57 expresses only a minimal amount of PAN. In cotransfection experiments, ORF57 alone increased PAN expression by 20-30-fold when compared to vector control. This accumulation function of ORF57 was dependent on a structured RNA element in the 5' PAN, named MRE (Mta responsive element), but not much so on an ENE (expression and nuclear retention element) in the 3' PAN previously reported by other studies. We showed that the major function of the 5' PAN MRE is increasing the RNA half-life of PAN in the presence of ORF57. Further mutational analyses revealed a core motif consisting of 9 nucleotides in the MRE-II , which is responsible for ORF57 interaction and function. The 9-nt core in the MRE-II also binds cellular PABPC1, but not the E1B-AP5 which binds another region of the MRE-II. In addition, we found that PAN RNA is partially exportable in the presence of ORF57. Together, our data provide compelling evidence as to how ORF57 functions to accumulate a non-coding viral RNA in the course of virus lytic infection.
KSHV; long non-coding RNA; ORF57; PAN; RNA stability; RNA accumulation; PABPC1; E1B-AP5
Human papillomavirus type 16 (HPV16) genome expresses six regulatory proteins (E1, E2, E4, E5, E6, and E7) which regulate viral DNA replication, gene expression, and cell function. We expressed HPV16 E2, E4, E6, and E7 from bacteria as GST fusion proteins and examined their possible functions in RNA splicing. Both HPV16 E2, a viral transactivator protein, and E6, a viral oncoprotein, inhibited splicing of pre-mRNAs containing an intron with suboptimal splice sites, whereas HPV5 E2 did not. The N-terminal half and the hinge region of HPV16 E2 as well as the N-terminal and central portions of HPV16 E6 are responsible for the suppression. HPV16 E2 interacts with pre-mRNAs through its C-terminal DNA-binding domain. HPV16 E6 binds pre-mRNAs via nuclear localization signal (NLS3) in its C-terminal half. Low-risk HPV6 E6, a cytoplasmic protein, does not bind RNA. Notably, both HPV16 E2 and E6 selectively bind to the intron region of pre-mRNAs and interact with a subset of cellular SR proteins. Together, these findings suggest that HPV16 E2 and E6 are RNA binding proteins and might play roles in posttranscriptional regulation during virus infection.
Human papillomavirus type 16; RNA splicing; RNA-protein interaction; SR proteins; Protein-protein interaction; viral proteins
Viral oncogenes are responsible for oncogenesis resulting from persistent virus infection. Although different human tumor viruses express different viral oncogenes and induce different tumors, their oncoproteins often target similar sets of cellular tumor suppressors or signal pathways to immortalize and/or transform infected cells. Expression of the viral E6 and E7 oncogenes in papillomavirus, E1A and E1B oncogenes in adenovirus, large T and small t antigen in polyomavirus, and Tax oncogene in HTLV-1 are regulated by alternative RNA splicing. However, this regulation is only partially understood. DNA tumor viruses also encode noncoding RNAs, including viral microRNAs, that disturb normal cell functions. Among the determined viral microRNA precursors, EBV encodes 25 from two major clusters (BART and BHRF1), KSHV encodes 12 from a latent region, human polyomavirus MCV produce only one microRNA from the late region antisense to early transcripts, but HPVs appears to produce no viral microRNAs.
Human papillomaviruses; Epstein-Barr virus; Kaposi sarcoma-associated herpesvirus; adenovirus; polyomavirus; human T-cell leukemia virus; viral noncoding RNA; viral microRNA; RNA splicing
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.
Cancer; splicing factors; SFRS3; SRp20; G2/M arrest; cell transformation; tumor induction
We identified a novel inhibitor of growth family member 2 (ING2) isoform, ING2b, which shares exon 2 with ING2a, but lacks the N-terminal p53 binding region. Contrary to ING2a, ING2b's promoter has no p53 binding sites. Consistently, activation of p53 led to suppression of ING2a, leaving ING2b unaffected. Through isoform-specific targeting, we showed that ING2a knockdown suppressed cell growth only in the presence of p53, ING2b knockdown had no effect on cell growth, and knockdown of both induced cell cycle arrest and apoptosis independently of p53. ING2a and ING2b have compensatory roles that protect cells from cell cycle arrest and apoptosis and may be involved in development of chemotherapeutic resistance.
ING2; ING2a; ING2b; p53; Isoform
In lower eukaryotes, Sir2 serves as a histone deacetylase and is implicated in chromatin silencing, longevity and genome stability. Here we mutated the SIRT1 gene, a homolog of yeast Sir2, in mice to study its function. We showed that a majority of SIRT1-null embryos died between E9.5–E14.5, displaying altered histone modification, impaired DNA damage response, and reduced ability to repair DNA damage. We demonstrated that SIRT1+/−;p53+/− mice developed tumors in multiple tissues, whereas activation of SIRT1 by resveratrol treatment reduced tumorigenesis. Finally, we showed that many human cancers exhibited reduced level of SIRT1 than their normal controls. Thus, SIRT1 acts as a tumor suppressor through its role in DNA damage response, genome integrity, and tumor suppression.
SIRT1 has diverse roles in various biological processes, including caloric restriction that causes changes in glucose metabolism and lifespan. The role of SIRT1 in cancer is currently under debate due to some recent different findings. It is known that calorie restriction, which activates SIRT1, extends lifespan and inhibits tumorigenesis. On the other hand, SIRT1 deacetylates p53 to decrease its activity. It was therefore hypothesized increased SIRT1 activity, although it extends lifespan, may elevate cancer risk. Here we demonstrate SIRT1 plays an important role in DNA damage response and genome integrity by maintaining proper chromatin structure and DNA damage repair foci formation. We further show that SIRT1 serves as a tumor suppressor in mice and in some types of human cancers.
γH2AX; Brca1; DNA damage repair; tumorigenesis