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Identification of Piwil2-like (PL2L) proteins that promote tumorigenesis.

Ye Y, Yin DT, Chen L, Zhou Q, Shen R, He G, Yan Q, Tong Z, Issekutz AC, Shapiro CL, Barsky SH, Lin H, Li JJ, Gao JX - PLoS ONE (2010)

Bottom Line: Consistently, PL2L proteins rather than Piwil2 are predominantly expressed in the cytoplasm or cytoplasm and nucleus of euchromatin-enriched tumor cells in human primary and metastatic cancers, such as breast and cervical cancers.Moreover, nuclear PL2L proteins are always co-expressed with nuclear NF-κB.These results reveal that PL2L60 can coordinate with NF-κB to promote tumorigenesis and might mediate a common pathway for tumor development without tissue restriction.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Ohio State University Medical Center, Columbus, Ohio, United States of America.

ABSTRACT
PIWIL2, a member of PIWI/AGO gene family, is expressed in the germline stem cells (GSCs) of testis for gametogenesis but not in adult somatic and stem cells. It has been implicated to play an important role in tumor development. We have previously reported that precancerous stem cells (pCSCs) constitutively express Piwil2 transcripts to promote their proliferation. Here we show that these transcripts de facto represent Piwil2-like (PL2L) proteins. We have identified several PL2L proteins including PL2L80, PL2L60, PL2L50 and PL2L40, using combined methods of Gene-Exon-Mapping Reverse Transcription Polymerase Chain Reaction (GEM RT-PCR), bioinformatics and a group of novel monoclonal antibodies. Among them, PL2L60 rather than Piwil2 and other PL2L proteins is predominantly expressed in various types of human and mouse tumor cells. It promotes tumor cell survival and proliferation in vitro through up-regulation of Stat3 and Bcl2 gene expressions, the cell cycle entry from G(0/1) into S-phase, and the nuclear expression of NF-κB, which contribute to the tumorigenicity of tumor cells in vivo. Consistently, PL2L proteins rather than Piwil2 are predominantly expressed in the cytoplasm or cytoplasm and nucleus of euchromatin-enriched tumor cells in human primary and metastatic cancers, such as breast and cervical cancers. Moreover, nuclear PL2L proteins are always co-expressed with nuclear NF-κB. These results reveal that PL2L60 can coordinate with NF-κB to promote tumorigenesis and might mediate a common pathway for tumor development without tissue restriction. The identification of PL2L proteins provides a novel insight into the mechanisms of cancer development as well as a novel bridge linking cancer diagnostics and anticancer drug development.

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Diagram of PIWIL2 and PL2L genes as well as their transcriptional and translational products.A, Schematic depiction of the genomic structure of PIWIL2 and PL2L genes. Five potential promoters inside the PWIL2 gene were identified. The promoter PPiwil2 is responsible for the transcription of Piwil2 mRNA in humans and mice, while other four promoters inside this gene, PPL2L60, PPL2L50, PPL2L42 and PPL2L40, may initiate the transcription of PL2L60, PL2L50, PL2L42 and PL2L40 genes, respectively. Human PL2L60, PL2L42 and PL2L40 mRNAs, and mouse PL2L50 mRNA have been identified in GenBank. The question marker “?” indicates that corresponding mRNA sequence has not been identified. B, Schematic presentation of mRNA structure of PIWIL2 and PL2L genes. All variants are truncated at 5′-end but all contain Piwil2 exons 15 to 23, except for PL2L40 which contains exons 13 to 20 and another two exons within the intron between exons 20 and 21 (red area in A & B). C, Schematic structures of Piwil2 and PL2L proteins. Compared to the full-length Piwil2 protein, all PL2L proteins are defective or absent of whole PAZ domain. Piwi domain is normal in PL2L60 but defective in PL2L50, PL2L40 and PL2L42.
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pone-0013406-g002: Diagram of PIWIL2 and PL2L genes as well as their transcriptional and translational products.A, Schematic depiction of the genomic structure of PIWIL2 and PL2L genes. Five potential promoters inside the PWIL2 gene were identified. The promoter PPiwil2 is responsible for the transcription of Piwil2 mRNA in humans and mice, while other four promoters inside this gene, PPL2L60, PPL2L50, PPL2L42 and PPL2L40, may initiate the transcription of PL2L60, PL2L50, PL2L42 and PL2L40 genes, respectively. Human PL2L60, PL2L42 and PL2L40 mRNAs, and mouse PL2L50 mRNA have been identified in GenBank. The question marker “?” indicates that corresponding mRNA sequence has not been identified. B, Schematic presentation of mRNA structure of PIWIL2 and PL2L genes. All variants are truncated at 5′-end but all contain Piwil2 exons 15 to 23, except for PL2L40 which contains exons 13 to 20 and another two exons within the intron between exons 20 and 21 (red area in A & B). C, Schematic structures of Piwil2 and PL2L proteins. Compared to the full-length Piwil2 protein, all PL2L proteins are defective or absent of whole PAZ domain. Piwi domain is normal in PL2L60 but defective in PL2L50, PL2L40 and PL2L42.

Mentions: Current data have shown that human PIWIL2 gene containing 23 exons is significantly larger than an average human gene (Fig. 2A). Different exon usage often leads to production of spliced mRNA variants, and differential promoter utilization inside a gene often results in the formation of related but functionally distinct proteins. These mechanisms greatly expand the coding capacity of a gene. Therefore, we reasoned that PIWIL2 gene might use these mechanisms to generate Piwil2 variants in particular conditions. To identify potential Piwil2 variants, we first used the software Gene2Promoter from Genomatix Software Inc. (Ann Arbor, MI) to analyze human and mouse PIWIL2 genes in order to find potential promoters inside these genes. The results showed that there are six potential promoters inside the PIWIL2 gene of humans or mice (Fig. 2A). Among these promoters, five promoters can be identified as the transcriptional initiators of PIWIL2, PL2L60, PL2L50, PL2L40 and PL2L42, respectively, in humans or mice (Fig. 2A). The promoter of PL2L60 is located in the region that starts from inside the intron 10 and includes most sequence of Exon 11 (Fig. 2A). This promoter initiates the transcription of PL2L60 mRNA that can be translated into 60 kDa protein PL2L60, in which PAZ domain is defective (Fig. 2B & 2C). The predicted PL2L60 mRNA is confirmed by transcribed sequence AK027497 from Genbank. The promoter of PL2L50 is located inside the intron 13 (Fig. 2A), which transcribe PL2L50 mRNA covering exons 14 to 23 (Fig. 2B) and its protein product contains Piwi domain truncated at N-terminus (Fig. 2C). The predicted PL2L50 is confirmed by transcribed sequence AK163647 (murine) from Genbank. The promoter of PL2L42 is located at the region that starts from inside the intron 14 and includes entire Exon 15 (Fig. 2A). This promoter initiates the transcription of PL2L42 mRNA that might be translated into a 42 kDa protein PL2L42, which is only a part of Piwi domain truncated at N-terminus (Fig. 2B & 2C). The predicted PL2L42 mRNA is supported by transcribed sequence AK001213 from Genbank. The promoter of PL2L40 is located inside the intron 12 (Fig. 2A). It initiates the transcription of PL2L40 gene, which contains exons from 13 to 20 and two additional exons within the intron between exons 20 and 21. The PL2L40 contains a C-terminus truncated Piwi domain (Fig. 2B & 2C). The predicted PL2L40 mRNA is confirmed by transcribed sequence XM_942053 in Genbank. These predicted mRNAs are all truncated at the 5′-end, resulting in a defect or absence of PAZ domain (Fig. 2C). The mRNA sequences of human PL2L60, PL2L50, PL2L40 and PL2L60 and murine PL2L50 can be found in Genbank.


Identification of Piwil2-like (PL2L) proteins that promote tumorigenesis.

Ye Y, Yin DT, Chen L, Zhou Q, Shen R, He G, Yan Q, Tong Z, Issekutz AC, Shapiro CL, Barsky SH, Lin H, Li JJ, Gao JX - PLoS ONE (2010)

Diagram of PIWIL2 and PL2L genes as well as their transcriptional and translational products.A, Schematic depiction of the genomic structure of PIWIL2 and PL2L genes. Five potential promoters inside the PWIL2 gene were identified. The promoter PPiwil2 is responsible for the transcription of Piwil2 mRNA in humans and mice, while other four promoters inside this gene, PPL2L60, PPL2L50, PPL2L42 and PPL2L40, may initiate the transcription of PL2L60, PL2L50, PL2L42 and PL2L40 genes, respectively. Human PL2L60, PL2L42 and PL2L40 mRNAs, and mouse PL2L50 mRNA have been identified in GenBank. The question marker “?” indicates that corresponding mRNA sequence has not been identified. B, Schematic presentation of mRNA structure of PIWIL2 and PL2L genes. All variants are truncated at 5′-end but all contain Piwil2 exons 15 to 23, except for PL2L40 which contains exons 13 to 20 and another two exons within the intron between exons 20 and 21 (red area in A & B). C, Schematic structures of Piwil2 and PL2L proteins. Compared to the full-length Piwil2 protein, all PL2L proteins are defective or absent of whole PAZ domain. Piwi domain is normal in PL2L60 but defective in PL2L50, PL2L40 and PL2L42.
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Related In: Results  -  Collection

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pone-0013406-g002: Diagram of PIWIL2 and PL2L genes as well as their transcriptional and translational products.A, Schematic depiction of the genomic structure of PIWIL2 and PL2L genes. Five potential promoters inside the PWIL2 gene were identified. The promoter PPiwil2 is responsible for the transcription of Piwil2 mRNA in humans and mice, while other four promoters inside this gene, PPL2L60, PPL2L50, PPL2L42 and PPL2L40, may initiate the transcription of PL2L60, PL2L50, PL2L42 and PL2L40 genes, respectively. Human PL2L60, PL2L42 and PL2L40 mRNAs, and mouse PL2L50 mRNA have been identified in GenBank. The question marker “?” indicates that corresponding mRNA sequence has not been identified. B, Schematic presentation of mRNA structure of PIWIL2 and PL2L genes. All variants are truncated at 5′-end but all contain Piwil2 exons 15 to 23, except for PL2L40 which contains exons 13 to 20 and another two exons within the intron between exons 20 and 21 (red area in A & B). C, Schematic structures of Piwil2 and PL2L proteins. Compared to the full-length Piwil2 protein, all PL2L proteins are defective or absent of whole PAZ domain. Piwi domain is normal in PL2L60 but defective in PL2L50, PL2L40 and PL2L42.
Mentions: Current data have shown that human PIWIL2 gene containing 23 exons is significantly larger than an average human gene (Fig. 2A). Different exon usage often leads to production of spliced mRNA variants, and differential promoter utilization inside a gene often results in the formation of related but functionally distinct proteins. These mechanisms greatly expand the coding capacity of a gene. Therefore, we reasoned that PIWIL2 gene might use these mechanisms to generate Piwil2 variants in particular conditions. To identify potential Piwil2 variants, we first used the software Gene2Promoter from Genomatix Software Inc. (Ann Arbor, MI) to analyze human and mouse PIWIL2 genes in order to find potential promoters inside these genes. The results showed that there are six potential promoters inside the PIWIL2 gene of humans or mice (Fig. 2A). Among these promoters, five promoters can be identified as the transcriptional initiators of PIWIL2, PL2L60, PL2L50, PL2L40 and PL2L42, respectively, in humans or mice (Fig. 2A). The promoter of PL2L60 is located in the region that starts from inside the intron 10 and includes most sequence of Exon 11 (Fig. 2A). This promoter initiates the transcription of PL2L60 mRNA that can be translated into 60 kDa protein PL2L60, in which PAZ domain is defective (Fig. 2B & 2C). The predicted PL2L60 mRNA is confirmed by transcribed sequence AK027497 from Genbank. The promoter of PL2L50 is located inside the intron 13 (Fig. 2A), which transcribe PL2L50 mRNA covering exons 14 to 23 (Fig. 2B) and its protein product contains Piwi domain truncated at N-terminus (Fig. 2C). The predicted PL2L50 is confirmed by transcribed sequence AK163647 (murine) from Genbank. The promoter of PL2L42 is located at the region that starts from inside the intron 14 and includes entire Exon 15 (Fig. 2A). This promoter initiates the transcription of PL2L42 mRNA that might be translated into a 42 kDa protein PL2L42, which is only a part of Piwi domain truncated at N-terminus (Fig. 2B & 2C). The predicted PL2L42 mRNA is supported by transcribed sequence AK001213 from Genbank. The promoter of PL2L40 is located inside the intron 12 (Fig. 2A). It initiates the transcription of PL2L40 gene, which contains exons from 13 to 20 and two additional exons within the intron between exons 20 and 21. The PL2L40 contains a C-terminus truncated Piwi domain (Fig. 2B & 2C). The predicted PL2L40 mRNA is confirmed by transcribed sequence XM_942053 in Genbank. These predicted mRNAs are all truncated at the 5′-end, resulting in a defect or absence of PAZ domain (Fig. 2C). The mRNA sequences of human PL2L60, PL2L50, PL2L40 and PL2L60 and murine PL2L50 can be found in Genbank.

Bottom Line: Consistently, PL2L proteins rather than Piwil2 are predominantly expressed in the cytoplasm or cytoplasm and nucleus of euchromatin-enriched tumor cells in human primary and metastatic cancers, such as breast and cervical cancers.Moreover, nuclear PL2L proteins are always co-expressed with nuclear NF-κB.These results reveal that PL2L60 can coordinate with NF-κB to promote tumorigenesis and might mediate a common pathway for tumor development without tissue restriction.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Ohio State University Medical Center, Columbus, Ohio, United States of America.

ABSTRACT
PIWIL2, a member of PIWI/AGO gene family, is expressed in the germline stem cells (GSCs) of testis for gametogenesis but not in adult somatic and stem cells. It has been implicated to play an important role in tumor development. We have previously reported that precancerous stem cells (pCSCs) constitutively express Piwil2 transcripts to promote their proliferation. Here we show that these transcripts de facto represent Piwil2-like (PL2L) proteins. We have identified several PL2L proteins including PL2L80, PL2L60, PL2L50 and PL2L40, using combined methods of Gene-Exon-Mapping Reverse Transcription Polymerase Chain Reaction (GEM RT-PCR), bioinformatics and a group of novel monoclonal antibodies. Among them, PL2L60 rather than Piwil2 and other PL2L proteins is predominantly expressed in various types of human and mouse tumor cells. It promotes tumor cell survival and proliferation in vitro through up-regulation of Stat3 and Bcl2 gene expressions, the cell cycle entry from G(0/1) into S-phase, and the nuclear expression of NF-κB, which contribute to the tumorigenicity of tumor cells in vivo. Consistently, PL2L proteins rather than Piwil2 are predominantly expressed in the cytoplasm or cytoplasm and nucleus of euchromatin-enriched tumor cells in human primary and metastatic cancers, such as breast and cervical cancers. Moreover, nuclear PL2L proteins are always co-expressed with nuclear NF-κB. These results reveal that PL2L60 can coordinate with NF-κB to promote tumorigenesis and might mediate a common pathway for tumor development without tissue restriction. The identification of PL2L proteins provides a novel insight into the mechanisms of cancer development as well as a novel bridge linking cancer diagnostics and anticancer drug development.

Show MeSH
Related in: MedlinePlus