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SC35 promotes splicing of the C5-V6-C6 isoform of CD44 pre-mRNA
LOH, TIING JEN,MOON, HEEGYUM,CHO, SUNGHEE,JUNG, DA-WOON,HONG, SEONG-EUI,KIM, DO HAN,GREEN, MICHAEL R.,ZHENG, XUEXIU,ZHOU, JIANHUA,SHEN, HAIHONG D.A. Spandidos 2014 ONCOLOGY REPORTS Vol.31 No.1
<P>CD44 is a cell membrane glycoprotein that mediates the response of cells to their cellular microenvironment and regulates growth, survival, differentiation and motility. CD44 pre-mRNA contains 20 exons, 10 of which are alternatively spliced. Among the CD44 spliced variants, one of the V6 exon-containing isoforms, the V4–7 variant which contains variable exons 4, 5, 6 and 7, confers metastatic potential to non-metastatic cells. However, the splicing regulation of the V6 exon is not completely understood. SC35 is an arginine-serine rich protein that regulates alternative splicing of various pre-mRNAs. In the present study, we established a stable cell line which indicates inclusion or skipping of the V6 exon with the RFP or GFP signal. Using this stable cell line, we found that the V6 exon and flanking introns of CD44 pre-mRNA contained SC35 response elements that regulate V6 splicing. RT-PCR analyses of the endogenous CD44 splicing showed that SC35 promotes the production of the C5-V6-C6 isoform. shRNA knockdown of SC35 showed that reduced expression of SC35 decreased expression of the V6 exon-containing isoforms. Our results reveal a novel mechanism of CD44V6 splicing.</P>
Loh, Tiing Jen,Moon, Heegyum,Cho, Sunghee,Jang, Hana,Liu, Yong Chao,Tai, Hongmei,Jung, Da-Woon,Williams, Darren R,Kim, Hey-Ran,Shin, Myung-Geun,Liao, D Joshua,Zhou, Jianhua,Shi, Wei,Zheng, Xuexiu,Shen National Hellenic Research Foundation 2015 ONCOLOGY REPORTS Vol.34 No.3
<P>CD44 is a transmembrane receptor for hyaluronic acid. CD44 pre-mRNA contains 19?exons, 9?of which are alternatively spliced. Among the CD44 spliced variants, the v4-7 variant, one of the v6?exon-containing isoforms that contains variable exon?4, 5, 6 and?7, confers metastatic potential to non-metastatic cells. Splicing of CD44 and the function of CD44 isoforms are different in breast cancer cells. hnRNP?A1 is a ubiquitously expressed protein with an inhibitory function in pre-mRNA splicing. We showed that CD44v6 isoform, which includes all of the v6-containing mRNA isoforms, had the highest expression level in non-metatatic breast cancer cells (MCF7) when compared to the level in metastatic breast cancer cells (MDA-MB-231) and normal breast cells (MCF10A). Furthermore we showed that hnRNP?A1 knockdown regulated splicing of CD44 differently in breast cancer cells. We showed here that CD44 isoform expression is completely different in MDA-MB-231 cells than that in MCF7 and MCF10A cells, whereas MCF7 and MCF10A cells had a similar expression pattern of CD44 isoforms. RT-PCR analysis of CD44v6 showed that MCF7 and MCF10A cells predominantly expressed the c5v6v7v8v9v10c6 isoform. However, in addition to this isoform, MDA-MB-231 cells also expressed the c5v6v8v9v10c6 and c5v6c6 isoforms. We also found that knockdown of hnRNP?A1 significantly reduced the expression of c5v6v7v8v9v10c6 and c5v6v8v9v10c6, and promoted the expression of c5v6c6. hnRNP?A1 knockdown significantly induced cell death. In addition, hnRNP?A1 knockdown induced a decrease in cell invasion in the MDA-MB-231 cells. Our results indicate that the knockdown of hnRNP A1 has a specific function on the splicing of CD44 in breast cancer cells.</P>
SR proteins regulate V6 exon splicing of CD44 pre-mRNA
( Tiing Jen Loh ),( Heegyum Moon ),( Ha Na Jang ),( Yongchao Liu ),( Namjeong Choi ),( Shengfu Shen ),( Darren Reece Williams ),( Da-woon Jung ),( Xuexiu Zheng ),( Haihong Shen ) 생화학분자생물학회(구 한국생화학분자생물학회) 2016 BMB Reports Vol.49 No.11
CD44 pre-mRNA includes 20 exons, of which exons 1-5 (C<sub>1</sub>-C<sub>5</sub>) and exons 16-20 (C<sub>6</sub>-C<sub>10</sub>) are constant exons, whereas exons 6-15 (V<sub>1</sub>-V<sub>10</sub>) are variant exons. V6-exon-containing isoforms have been known to be implicated in tumor cell invasion and metastasis. In the present study, we performed a SR protein screen for CD44 V<sub>6</sub> splicing using overexpression and lentivirus-mediated shRNA treatment. Using a CD44 V<sub>6</sub> minigene, we demonstrate that increased SRSF3 and SRSF4 expression do not affect V<sub>6</sub> splicing, but increased expression of SRSF1, SRSF6 and SRSF9 significantly inhibit V<sub>6</sub> splicing. In addition, using a constitutive exon-specific primer set, we could not detect alterations of CD44 splicing after SR protein-targeting shRNA treatment. However, using a V<sub>6</sub> specific primer, we identified that reduced SRSF2 expression significantly reduced the V6 isoform, but increased V<sub>6-10</sub> and V<sub>6,8-10</sub> isoforms. Our results indicate that SR proteins are important regulatory proteins for CD44 V<sub>6</sub> splicing. [BMB Reports 2016; 49(11): 612-616]
CRISPR as a strong gene editing tool
( Shengfu Shen ),( Tiing Jen Loh ),( Hongling Shen ),( Xuexiu Zheng ),( Haihong Shen ) 생화학분자생물학회(구 한국생화학분자생물학회) 2017 BMB Reports Vol.50 No.1
Clustered regularly-interspaced short palindromic repeats (CRISPR) is a new and effective genetic editing tool. CRISPR was initially found in bacteria to protect it from virus invasions. In the first step, specific DNA strands of virus are identified by guide RNA that is composed of crRNA and tracrRNA. Then RNAse III is required for producing crRNA from pre-crRNA. In The second step, a crRNA:tracrRNA:Cas9 complex guides RNase III to cleave target DNA. After cleavage of DNA by CRISPR-Cas9, DNA can be fixed by Non- Homologous End Joining (NHEJ) and Homology Directed Repair (HDR). Whereas NHEJ is simple and random, HDR is much more complex and accurate. Gene editing by CRISPR is able to be applied to various biological field such as agriculture and treating genetic diseases in human. [BMB Reports 2017; 50(1): 20-24]
Splicing inhibition of U2AF<sup>65</sup> leads to alternative exon skipping
Cho, Sunghee,Moon, Heegyum,Loh, Tiing Jen,Jang, Ha Na,Liu, Yongchao,Zhou, Jianhua,Ohn, Takbum,Zheng, Xuexiu,Shen, Haihong National Academy of Sciences 2015 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.112 No.32
<P><B>Significance</B></P><P>Transcription is a biological procedure in which DNA is transcribed to an RNA molecule. However, only fragments of this RNA are needed for protein synthesis. These fragments are exons that are interrupted by introns. Introns are removed by so-called RNA splicing process. Some exons could be alternatively included or excluded from the final RNA molecule. In this study, we have found that U2 snRNP auxiliary factor 65 kDa (U2AF<SUP>65</SUP>), a general splicing regulator, can significantly promote the exclusion of alternative exons. Strikingly, U2AF<SUP>65</SUP> suppresses flanking intron splicing of alternative exons, and even constitutive intron splicing. We deduce that the stimulatory effects of U2AF<SUP>65</SUP> on alternative exon exclusion are induced by the splicing inhibitory effects of U2AF<SUP>65</SUP>.</P><P>U2 snRNP auxiliary factor 65 kDa (U2AF<SUP>65</SUP>) is a general splicing factor that contacts polypyrimidine (Py) tract and promotes prespliceosome assembly. In this report, we show that U2AF<SUP>65</SUP> stimulates alternative exon skipping in spinal muscular atrophy (SMA)-related survival motor neuron (<I>SMN</I>) pre-mRNA. A stronger 5′ splice-site mutation of alternative exon abolishes the stimulatory effects of U2AF<SUP>65</SUP>. U2AF<SUP>65</SUP> overexpression promotes its own binding only on the weaker, not the stronger, Py tract. We further demonstrate that U2AF<SUP>65</SUP> inhibits splicing of flanking introns of alternative exon in both three-exon and two-exon contexts. Similar U2AF<SUP>65</SUP> effects were observed in Fas (Apo-1/CD95) pre-mRNA. Strikingly, we demonstrate that U2AF<SUP>65</SUP> even inhibits general splicing of adenovirus major late (Ad ML) or β-globin pre-mRNA. Thus, we conclude that U2AF<SUP>65</SUP> possesses a splicing Inhibitory function that leads to alternative exon skipping.</P>
SRSF2 directly inhibits intron splicing to suppresses cassette exon inclusion
( Heegyum Moon ),( Sunghee Cho ),( Tiing Jen Loh ),( Ha Na Jang ),( Yongchao Liu ),( Namjeong Choi ),( Jagyeong Oh ),( Jiyeon Ha ),( Jianhua Zhou ),( Sungchan Cho ),( Dong-eun Kim ),( Michael B. Ye ) 생화학분자생물학회(구 한국생화학분자생물학회) 2017 BMB Reports Vol.50 No.8
SRSF2, a Serine-Arginine rich (SR) protein, is a splicing activator that mediates exon inclusion and exclusion events equally well. Here we show SRSF2 directly suppresses intron splicing to suppress cassette exon inclusion in SMN pre-mRNA. Through a serial mutagenesis, we demonstrate that a 10 nt RNA sequence surrounding the branch-point (BP), is important for SRSF2-mediated inhibition of cassette exon inclusion through directly interacting with SRSF2. We conclude that SRSF2 inhibits intron splicing to promote exon exclusion. [BMB Reports 2017; 50(8): 423-428]
Cho, Sunghee,Moon, Heegyum,Loh, Tiing Jen,Oh, Hyun Kyung,Kim, Hey-Ran,Shin, Myung-Geun,Liao, D. Joshua,Zhou, Jianhua,Zheng, Xuexiu,Shen, Haihong Hindawi Publishing Corporation 2014 The Scientific World Journal Vol.2014 No.-
<P>Spinal muscular atrophy (SMA) is a human genetic disease which occurs because of the deletion or mutation of SMN1 gene. SMN1 gene encodes the SMN protein which plays a key role in spliceosome assembly. Although human patients contain SMN2, a duplicate of SMN1, splicing of SMN2 produces predominantly exon 7 skipped isoform. In order to understand the functions of splice site sequences on exon 7 and 8, we analyzed the effects of conserved splice site sequences on exon 7 skipping of SMN2 and SMN1 pre-mRNA. We show here that conserved 5′ splice site sequence of exon 7 promoted splicing of nearby exons and subsequently reduced splicing of distant exons. However, to our surprise, conserved 3′ splice site sequence of exon 7 and 8 did not promote splicing of nearby exons. By contrast, the mutation inhibited splicing of nearby exons and subsequently promoted splicing of distant exons. Our study shows that 3′ splice sites of exon 7 and 8 contain enhancer for their splice site selection, in addition to providing cleavage sites.</P>
Zheng, Xuexiu,Cho, Sunghee,Moon, Heegyum,Loh, Tiing Jen,Oh, Huyn Kyung,Green, Michael R.,Shen, Haihong Cold Spring Harbor Laboratory Press 2014 RNA Vol.20 No.4
<P>This study provides new insight into how the immunoglobulin splicing inhibitor sequence blocks splicing. Evidence indicates the polypyrimidine tract binding protein bound to the splicing inhibitor element prevents U2 snRNA from base-pairing to the branch point.</P>
Identification of novel splicing variants from RON proto-oncogene pre-mRNA.
Moon, Heegyum,Cho, Sunghee,Yang, Xiaoming,Zhou, Jianhua,Loh, Tiing Jen,Zheng, Xuexiu,Shen, Haihong National Hellenic Research Foundation 2012 ONCOLOGY REPORTS Vol.28 No.6
<P>RON is a proto-oncogene that induces cell dissociation, migration and matrix invasion. RON?160, a splicing variant of RON, is a natural splicing product in colon cancers that is produced through skipping of exons 5 and 6 in alternative splicing process. RON?160 promotes cellular transformation in?vitro and tumor formation in vivo. We present, here, two novel splicing variants of RON in the partial splicing events that involve exons 5 and 6. The common facts of these two novel splicing variants are that exons 4-7 are included. In addition, intron 4 is spliced whereas intron 5 is included in both variants. The difference of these two isoforms is the inclusion or skipping of intron 6. In one variant intron 6 is included, but intron 6 is skipped in another variant. These two variants should be truncated but these proteins have not yet been detected.</P>