open access

Vol 57, No 2 (2019)
ORIGINAL PAPERS
Published online: 2019-05-20
Submitted: 2019-01-02
Accepted: 2019-04-12
Get Citation

L1-ORF1p and Ago2 are involved in a siRNA-mediated regulation for promoter activity of L1-5’UTR

Liu Shi, Huanhuan Shi, Liangliang Lou, Xiaoning Yuan, Yunfeng Zhu
DOI: 10.5603/FHC.a2019.0006
·
Pubmed: 31112282
·
Folia Histochem Cytobiol 2019;57(2):56-63.

open access

Vol 57, No 2 (2019)
ORIGINAL PAPERS
Published online: 2019-05-20
Submitted: 2019-01-02
Accepted: 2019-04-12

Abstract

Introduction. Long interspersed nuclear elements-1 (L1), as the only one self-active retrotransposon of the mobile element, was found to be generally activated in tumor cells. The 5‘UTR of L1 (L1-5’UTR) contains both sense and antisense bidirectional promoters, transcription products of which can generate double-strand RNA (dsRNA). In addition, L1-ORF1p, a dsRNA binding protein encoded by L1, is considered to engage in some RNA-protein (RNP) formation. Ago2, one of the RISC components, can bind to dsRNA to form RISC complex, but its role in L1 regulation still remains unclear. Due that the 5‘UTR of L1 (L1-5’UTR) contains both sense and antisense bidirectional promoters, so the activities in both string were identified. A dsRNA-mediated regulation of L1-5’UTR, with the feedback regulation of L1-ORF1p as well as other key molecules engaged (Ago1–4) in this process, was also investigated.

Material and methods. Genomic DNA was extracted from HEK293 cells and subjected to L1-5’UTR prepa­ration by PCR. Report gene system pIRESneo with SV40 promoter was employed. The promoter activities of different regions in L1-5’UTR were identified by constructing these regions into pIRESneo, which SV40 region was removed prior, to generate different recombinant plasmids. The promoter activities in recombinant plasmids were detected by the luciferase expression assay. Western blot and co-immunoprecipitation were employed to identify proteins expression and protein-protein interaction respectively.

Results. Ago2 is a member of Agos family, which usually forms a RISC complex with si/miRNA and is involved in post- transcriptional regulation of many genes. Here L1-ORF1p and Ago2 conducts a regulation as a negative feedback for L1-5'UTR sense promoter. L1-ORF1p could form the immune complexes with Ago1, Ago2 and Ago4, respectively.

Conclusions. L1-5’UTR harbors both sense and antisense promoter activity and a dsRNA-mediated regulation is responsible for L1-5’UTR regulation. Agos proteins and L1-ORF1p were engaged in this process.

Abstract

Introduction. Long interspersed nuclear elements-1 (L1), as the only one self-active retrotransposon of the mobile element, was found to be generally activated in tumor cells. The 5‘UTR of L1 (L1-5’UTR) contains both sense and antisense bidirectional promoters, transcription products of which can generate double-strand RNA (dsRNA). In addition, L1-ORF1p, a dsRNA binding protein encoded by L1, is considered to engage in some RNA-protein (RNP) formation. Ago2, one of the RISC components, can bind to dsRNA to form RISC complex, but its role in L1 regulation still remains unclear. Due that the 5‘UTR of L1 (L1-5’UTR) contains both sense and antisense bidirectional promoters, so the activities in both string were identified. A dsRNA-mediated regulation of L1-5’UTR, with the feedback regulation of L1-ORF1p as well as other key molecules engaged (Ago1–4) in this process, was also investigated.

Material and methods. Genomic DNA was extracted from HEK293 cells and subjected to L1-5’UTR prepa­ration by PCR. Report gene system pIRESneo with SV40 promoter was employed. The promoter activities of different regions in L1-5’UTR were identified by constructing these regions into pIRESneo, which SV40 region was removed prior, to generate different recombinant plasmids. The promoter activities in recombinant plasmids were detected by the luciferase expression assay. Western blot and co-immunoprecipitation were employed to identify proteins expression and protein-protein interaction respectively.

Results. Ago2 is a member of Agos family, which usually forms a RISC complex with si/miRNA and is involved in post- transcriptional regulation of many genes. Here L1-ORF1p and Ago2 conducts a regulation as a negative feedback for L1-5'UTR sense promoter. L1-ORF1p could form the immune complexes with Ago1, Ago2 and Ago4, respectively.

Conclusions. L1-5’UTR harbors both sense and antisense promoter activity and a dsRNA-mediated regulation is responsible for L1-5’UTR regulation. Agos proteins and L1-ORF1p were engaged in this process.

Get Citation

Keywords

L1-5’UTR; Ago2; L1-ORF1p; antisense promoter; siRNA; HEK293 cells; protein interactions

About this article
Title

L1-ORF1p and Ago2 are involved in a siRNA-mediated regulation for promoter activity of L1-5’UTR

Journal

Folia Histochemica et Cytobiologica

Issue

Vol 57, No 2 (2019)

Pages

56-63

Published online

2019-05-20

DOI

10.5603/FHC.a2019.0006

Pubmed

31112282

Bibliographic record

Folia Histochem Cytobiol 2019;57(2):56-63.

Keywords

L1-5’UTR
Ago2
L1-ORF1p
antisense promoter
siRNA
HEK293 cells
protein interactions

Authors

Liu Shi
Huanhuan Shi
Liangliang Lou
Xiaoning Yuan
Yunfeng Zhu

References (21)
  1. Baillie JK, Barnett MW, Upton KR, et al. Somatic retrotransposition alters the genetic landscape of the human brain. Nature. 2011; 479(7374): 534–537.
  2. Denli AM, Narvaiza I, Kerman BE, et al. Primate-specific ORF0 contributes to retrotransposon-mediated diversity. Cell. 2015; 163(3): 583–593.
  3. Pittoggi C, Sciamanna I, Mattei E, et al. Role of endogenous reverse transcriptase in murine early embryo development. Mol Reprod Dev. 2003; 66(3): 225–236.
  4. Macia A, Muñoz-Lopez M, Cortes JL, et al. Epigenetic control of retrotransposon expression in human embryonic stem cells. Mol Cell Biol. 2011; 31(2): 300–316.
  5. Wanichnopparat W, Suwanwongse K, Pin-On P, et al. Genes associated with the cis-regulatory functions of intragenic LINE-1 elements. BMC Genomics. 2013; 14: 205.
  6. Morales JF, Snow ET, Murnane JP. Environmental factors affecting transcription of the human L1 retrotransposon. II. Stressors. Mutagenesis. 2003; 18(2): 151–158.
  7. Hansen RS. X inactivation-specific methylation of LINE-1 elements by DNMT3B: implications for the Lyon repeat hypothesis. Hum Mol Genet. 2003; 12(19): 2559–2567.
  8. Takai D, Yagi Y, Habib N, et al. Hypomethylation of LINE1 retrotransposon in human hepatocellular carcinomas, but not in surrounding liver cirrhosis. Jpn J Clin Oncol. 2000; 30(7): 306–309.
  9. Jürgens B, Schmitz-Dräger BJ, Schulz WA. Hypomethylation of L1 LINE sequences prevailing in human urothelial carcinoma. Cancer Res. 1996; 56(24): 5698–5703.
  10. Kitkumthorn N, Mutirangura A. Long interspersed nuclear element-1 hypomethylation in cancer: biology and clinical applications. Clin Epigenetics. 2011; 2(2): 315–330.
  11. Aschacher T, Wolf B, Enzmann F, et al. LINE-1 induces hTERT and ensures telomere maintenance in tumour cell lines. Oncogene. 2016; 35(1): 94–104.
  12. Reyes-Reyes EM, Aispuro I, Tavera-Garcia MA, et al. LINE-1 couples EMT programming with acquisition of oncogenic phenotypes in human bronchial epithelial cells. Oncotarget. 2017; 8(61): 103828–103842.
  13. Martin SL. The ORF1 protein encoded by LINE-1: structure and function during L1 retrotransposition. J Biomed Biotechnol. 2006; 2006(1): 45621.
  14. Goodier JL, Zhang L, Vetter MR, et al. LINE-1 ORF1 protein localizes in stress granules with other RNA-binding proteins, including components of RNA interference RNA-induced silencing complex. Mol Cell Biol. 2007; 27(18): 6469–6483.
  15. Martin SL, Bushman FD. Nucleic acid chaperone activity of the ORF1 protein from the mouse LINE-1 retrotransposon. Mol Cell Biol. 2001; 21(2): 467–475.
  16. Swergold GD. Identification, characterization, and cell specificity of a human LINE-1 promoter. Mol Cell Biol. 1990; 10(12): 6718–6729.
  17. Mätlik K, Redik K, Speek M. L1 antisense promoter drives tissue-specific transcription of human genes. J Biomed Biotechnol. 2006; 2006(1): 71753.
  18. Deng Y, Wang CC, Choy KW, et al. Therapeutic potentials of gene silencing by RNA interference: principles, challenges, and new strategies. Gene. 2014; 538(2): 217–227.
  19. Horman SR, Svoboda P, Luning Prak ET. The potential regulation of L1 mobility by RNA interference. J Biomed Biotechnol. 2006; 2006(1): 32713.
  20. Aporntewan C, Phokaew C, Piriyapongsa J, et al. Hypomethylation of intragenic LINE-1 represses transcription in cancer cells through AGO2. PLoS One. 2011; 6(3): e17934.
  21. Sheu-Gruttadauria J, MacRae IJ. Structural Foundations of RNA Silencing by Argonaute. J Mol Biol. 2017; 429(17): 2619–2639.

Important: This website uses cookies. More >>

The cookies allow us to identify your computer and find out details about your last visit. They remembering whether you've visited the site before, so that you remain logged in - or to help us work out how many new website visitors we get each month. Most internet browsers accept cookies automatically, but you can change the settings of your browser to erase cookies or prevent automatic acceptance if you prefer.

By "Via Medica sp. z o.o." sp.k., ul. Świętokrzyska 73, 80–180 Gdańsk

tel.:+48 58 320 94 94, faks:+48 58 320 94 60, e-mail:  viamedica@viamedica.pl