A Novel Long Non-Coding RNA, SNHG4 Complex With Eukaryotic Initiation Factor-4E and Regulate Aberrant Protein Translation In Mantle Cell Lymphoma: Implications For Novel Biomarker

Long noncoding RNAs (lncRNAs) are defined as RNA-like transcripts that are over 200 nucleotides and lack significant open reading frames. Some lncRNAs such as HOTAIR, MALAT1 and H19 have been found to be associated with clinical prognosis and are potential drivers of cancer progression in cancers of the breast, lung, and liver respectively. The role of lncRNAs in lymphoma is unknown. Dysregulation of eIF4E (a key component of the translation initiation complex eIF4F) influences global protein translation, especially the translation of “weak” mRNAs that can be malignancy-related. We and others have found that eIF4E is dysregulated in B-cell lymphoma. The aim of this study is to identify eIF4E-associated lncRNAs through next generation RNA-Sequencing (NGS RNA-Seq) and delineate their role in protein translation in lymphoma.

RNA-immunoprecipitation (RNA-IP) was used to pull down eIF4E-bound lncRNA in lymphoma cells. eIF4E-bound lncRNAs were immunoprecipitated with eIF4E antibody or IgG control in Jeko, a mantle cell lymphoma (MCL) cell line and sent for microarray analysis and NGS-RNA-Seq for identification of lncRNAs. The microarray analysis showed that several lncRNAs were enriched with eIF4E antibody compared to IgG control. These included SNHG4 (13.6 fold), SNHG12 (4.8 fold), NCRNA00171 (4.8 fold) and IPW (4.6 fold), GNASAS (3.5 fold), SNHG7 (3.3 fold), NCRNA00182 (2.7 fold), NCRNA00094 (2.6 fold), NCRNA00188 (2.4 fold) and NCRNA00201 (2.1 fold). The binding of these lncRNAs to eIF4E was further confirmed by RT-PCR in Jeko, Mino and Granta MCL cell lines. Next, we looked the expression of these lncRNAs by qRT- PCR in the MCL cell lines and normal controls. We found SNHG4 and IPW to be overexpressed in all the MCL cell lines, while SNHG12 and NCRNA00201 were overexpressed in the selected cell lines. No significant difference was found for the expression of NCRNA00171 and NCRNA00182 in any of the MCL cell lines compared to controls. Overall, these data suggest that several lncRNA have altered expression in malignant B-cells. Considering that the microarray assay only covered a limited number of lncRNAs, we further confirmed eIF4E bound lncRNA by NGS RNA-Seq in Jeko MCL and normal control. The binding of 10/13 lncRNA mentioned above with eIF4E were found upregulated by NGS-RNA-Seq. In addition several novel lncRNAs such as SNHG1 (161.6), AC091814.2 (98.8) and RP11-304L19.5 (64.2) showed up in NGS-RNA-Seq data. These data suggest that lncRNAs, such as SNHG12, SNHG4, and SNHG1 bind to eIF4E with high affinity in malignant B-cells and might play a role in protein translation. We knocked down the expression of SNHG4 through siRNA and demonstrated that cell proliferation and global protein translation was inhibited in lymphoma cells. To further confirm the role of SNHG4 in translation regulation, a plasmid, which contains a renilla luciferase driven by SV40 promoter, was co-transfected with SNHG4 siRNA into Mino cells. The luciferase signal, decreased compared with the cells transfected with nontargeting siRNA. These data suggest that SNHG4 is involved in the regulation of protein translation. In order to clarify the mechanism of lncRNAs bound to eIF4E we searched for RNA binding sites or motifs in eIF4E protein using the web-based tools, BindN and PPRInt. Interestingly two RNA binding motifs, KNKRGGRWLITLNKQQRRS and SHADTATKSGSTTKNR, were found in eIF4E based on the prediction. To examine whether lncRNAs bind with eIF4E through these RNA binding motifs, an eIF4E mutant plasmid with both RNA binding motifs deleted (eIF4E^Del), was constructed and transfected transiently into HEK-293T cells along with eIF4E^WT plasmid. RNA-IP data showed that the lncRNAs SNHG12, SNHG4 and SNHG1 were not able to bind with eIF4E in eIF4E^Del-transfected cells compared with that of eIF4E^WT, suggesting that these lncRNAs complex with eIF4E through RNA-binding motifs within the eIF4E.

Overall, our results show that the lncRNAs, SNHG1 and SNHG4 are able to bind with eIF4E and regulate protein translation. Since lncRNAs had been found to play roles in the regulation of gene expression, including transcription, splicing and mRNA stability, our results may broaden the view of the functional role of lncRNAs in translation in lymphoma cells and in other cancers. Furthermore, our results also suggested that SNHG4 lncRNAs might be served as potential biomarkers for MCL and other B cell lymphomas for translation therapy.