Abstract
The multifunctional protein Transglutaminase type 2, is associated with cancer epithelial mesenchymal transition, invasiveness, stemness and drugs resistance. Several variant isoforms and non-coding RNAs are present in cancer and this report explored the expression of these transcripts of the TGM2 gene in cancer cell lines after induction with all-trans retinoic acid. The expression of truncated variants along with two long non-coding RNAs, was demonstrated. One of these is coded from the first intron and the Last Exon Variant is constituted by a sequence corresponding to the last three exons and the 3′UTR. Analysis of ChIP-seq data, from ENCODE project, highlighted factors interacting with intronic sequences, which could interfere with the progression of RNApol II at checkpoints, during the elongation process. Some relevant transcription factors, bound in an ATRA-dependent way, were found by RNA immunoprecipitation, notably GATA3 mainly enriched to Last Exon Variant non-coding RNA. The involvement of NMD in the regulation of the ratio among these transcripts was observed, as the prevalent recovering of Last Exon Variant to phUPF1-complexes, with decrease of the binding towards other selective targets. This study contributes to identify molecular mechanisms regulating the ratio among the variants and improves the knowledge about regulatory roles of the non-coding RNAs of the TGM2 gene.
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Abbreviations
- AAs:
-
Amino acids
- Abs:
-
Antibodies
- APL:
-
Acute promyelocytic leukaemia
- AS:
-
Antisense oligonucleotide
- ATRA:
-
All-trans retinoic acid
- CTCF:
-
CCCTC-binding factor
- FAB:
-
French–American–British
- FBS:
-
Foetal bovine serum
- GEO:
-
Gene expression omnibus
- HDACs:
-
Histone deacetylases
- HPRT1:
-
Hypoxanthine phosphoribosyltransferase 1 gene
- LEV:
-
Last Exon Variant
- lncRNA:
-
Long non-coding RNA
- miRNAs:
-
microRNAs
- NI:
-
Normalised signal intensity
- NMD:
-
Nonsense-mediated mRNA decay
- PBS:
-
Phosphate-buffered saline
- phUPF1:
-
Phosphorylated ATP-dependent RNA helicase upframeshift 1
- RA:
-
Retinoic acid
- RARα:
-
Retinoic acid receptor α
- RAREs:
-
Retinoic acid responsive elements
- RIP:
-
RNA immunoprecipitation
- RRE:
-
Retinoid receptor element
- RT-qPCR:
-
Reverse transcription and real time quantitative PCR
- RXR α:
-
Retinoid Receptor X α
- TGM2 :
-
Transglutaminase type 2 gene
- TG2:
-
Transglutaminase type 2
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Acknowledgements
The cell lines employed in this study were kind gifts from Professors Paola Secchiero (HL-60 and NB4 cells), Michael U. Martin (SK-N-SH cells) and Ralf Hass (SH-SY5Y cells). The authors thank Prof. Giovanni Bernacchia for having made available the instrument to perform qPCR reactions. We are grateful to Prof. Cristian Taccioli and Dr. Marco Ricci for their advice and valuable help to address the study of TG2-lncRNA molecules. Finally, we thank Co.Pe.Go. (Soc.Coop.O.P.) for liberal voluntary contribution to the present research.
Funding
The research was supported by Grants FIR2017 (Unife) and FAR2018 (Unife) to C.M.B. and by Associazione Italiana Ricerca sul Cancro AIRC (IG 17063) to S.V.
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Methodology and analysis, OF, FC, GA, CC, and NB; biostatistical analysis: LM; devising of the study, CMB and NB; writing, OF, CF, CMB and NB; supervision, SV, CMB and NB; funding acquisition, CMB and SV.
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726_2019_2766_MOESM1_ESM.pptx
Supplementary Fig. 1 Drawing describing the secondary RNA structure of the TG2-lncRNA with base-paired regions generated using Vienna web RNA service considering two approaches for the analysis: a to have the structure with less free-energy structure, b to employ a model based on centroids. In the colour bar, red corresponds to high and blue to a low probability that the structure is hired (PPTX 249 kb)
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Franzese, O., Minotti, L., Aguiari, G. et al. Involvement of non-coding RNAs and transcription factors in the induction of Transglutaminase isoforms by ATRA. Amino Acids 51, 1273–1288 (2019). https://doi.org/10.1007/s00726-019-02766-7
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DOI: https://doi.org/10.1007/s00726-019-02766-7