Nuclear retention element recruits U1 snRNP components to restrain spliced lncRNAs in the nucleus

Abstract

In contrast to cytoplasmic localization of spliced mRNAs, many spliced lncRNAs are localized in the nucleus. To investigate the mechanism, we used lncRNA MEG3 as a reporter and mapped a potent nuclear retention element (NRE), deletion of this element led to striking export of MEG3 from the nucleus to the cytoplasm. Insertion of the NRE resulted in nuclear retention of spliced lncRNA as well as spliced mRNA. We further purified RNP assembled on the NRE in vitro and identified the proteins by mass spectrometry. Screen using siRNA revealed depletion of U1 snRNP components SNRPA, SNRNP70 or SNRPD2 caused significant cytoplasmic localization of MEG3 reporter transcripts. Co-knockdown these factors in HFF1 cells resulted in an increased cytoplasmic distribution of endogenous lncRNAs. Together, these data support a model that U1 snRNP components restrain spliced lncRNAs in the nucleus via the interaction with nuclear retention element.

Keywords: LncRNA localization; MEG3; U1 snRNP components; nuclear retention element.

Figure 1.

Subcellular localization of MEG3 reporter transcripts. A. Schematic of MEG3 genomic structure, MEG3 reporter and ANCR reporter. Introns were showed as a solid line and exons were showed as boxes. For MEG3 gene and ANCR reporter construct, the numbers ontop and beneath indicated the length of each introns and exons, respectively, for MEG3 reporter construct, the number beneath indicated the length of MEG3 cDNA used in this study. B. Western blot to show the efficiency of nucleus/cytoplasm separation. Cyt: cytoplasm; Nuc: nucleus. C. RT-PCR to show that MEG3 reporter transcripts enriched in the nucleus and ANCR reporter transcripts enriched in the cytoplasm. RT: reverse transcriptase. D. RNA-FISH showing nuclear localization of MEG3 and subcellular localization of ANCR after transient transfection.

Figure 2.

Mapping nuclear retention element in MEG3. A. Schematic of MEG3 reporter and RNA-FISH to show subcellular localization of MEG3 transcripts after transient transfection to HeLa cells. B-I. Schematic of MEG3 deletion constructs and RNA-FISH to show subcellular localization of MEG3 transcripts. Dotted line: MEG3 sequence deleted; the numbers beneath indicated the starting and ending position of MEG3 cDNA in the specific construct; dots on top of MEG3 boxes in F&G: two point mutations introduced to eliminate cryptic splicing (See Suppl Fig. S3).

Figure 3.

Insertion of nuclear retention element enriched cytoplasmic RNA in the nucleus. A. Schematic of chimeric NRE-ANCR and NRE-β-globin WT constructs. The numbers indicated the length of NRE, each intron and exon. Dots on top of NRE indicated 2-point mutations introduced. T7 and R indicated the positions of primers used for amplification in B. B. RT-PCR showing correct splicing after insertion of NRE. C. RNA-FISH showing subcellular localization of the chimeric transcripts.

Figure 4.

Depletion of SNRPA/SNRNP70/SNRPD2 led to cytoplasmic accumulation of MEG3 reporter transcripts and enrichment of endogenous lncRNAs in cytoplasm. A. Western blot showing knockdown efficiency after treatment of HeLa cells with specific siRNA. B. RNA-FISH showing depletion of SNRPA/SNRNP70/SNRPD2 resulted in shift of MEG3 reporter transcripts from nucleus to cytoplasm. C. The percentage of cells with apparent FISH signal in cytoplasm increased significantly after depletion of SNRPA/SNRNP70/SNRPD2. (n = 3) ***: p < 0.001. D. RT-PCR results showing elevated distribution of MEG3 reporter transcripts in cytoplasm after depletion of SNRPA/SNRNP70/SNRPD2. (n = 3) *: p < 0.05; **: p < 0.01; ***: p < 0.001. E. Western blot showing knockdown efficiency after treatment of HFF1 with siRNAs against SNRPA/SNRNP70/SNRPD2 simultaneously. Si-3KD: triple knockdown of SNRPA, SNRNP70 and SNRPD2. F. RT-PCR showing distribution of endogenous lncRNAs in cytoplasm and nucleus after siRNA treatment of HFF1 cells. C: cytoplasm; N: nucleus; W: whole cell. G. Quantification of lncRNAs localized in the cytoplasm. (n = 3) *: p < 0.05; **: p < 0.01; ***: p < 0.001.