Intronless mRNAs transit through nuclear speckles to gain export competence

Abstract

Nuclear speckles (NSs) serve as splicing factor storage sites. In this study, we unexpectedly found that many endogenous intronless mRNAs, which do not undergo splicing, associate with NSs. These associations do not require transcription, polyadenylation, or the polyA tail. Rather, exonic splicing enhancers present in intronless mRNAs and their binding partners, SR proteins, promote intronless mRNA localization to NSs. Significantly, speckle targeting of mRNAs promotes the recruitment of the TREX export complex and their TREX-dependent nuclear export. Furthermore, TREX, which accumulates in NSs, is required for releasing intronless mRNAs from NSs, whereas NXF1, which is mainly detected at nuclear pores, is not. Upon NXF1 depletion, the TREX protein UAP56 loses speckle concentration but coaccumulates with intronless mRNAs and polyA RNAs in the nucleoplasm, and these RNAs are trapped in NSs upon UAP56 codepletion. We propose that the export-competent messenger RNP assembly mainly occurs in NSs for intronless mRNAs and that entering NSs serves as a quality control step in mRNA export.

Figure 1.

Exogenously expressed naturally intronless mRNAs associate with NSs. (A) Top: Schematic of reporter constructs. Sequences from the vector and the genes of interest are indicated as a cyan line and gray bar, respectively. The positions of the promoter, polyA (pA) site, and FISH probe are indicated. Bottom: Equal amounts of reporter constructs were microinjected into HeLa nuclei, and α-amanitin (4 µg/ml) was added 20 min after injection. At 30 min (cG and wG), 1 h (CJUN and JUND), 2 h (HSPA1A), or 4 h (others) after the addition of α-amanitin, FISH with the 3′ vector probe and IF with the SRSF2 antibody were performed. Higher magnification of the boxed regions is shown. The red and green lines in the graphs show the intensity of the FISH and SRSF2 IF signal along the freely positioned arrow indicated from A to B, respectively. CMV, cytomegalovirus; CDS, coding sequence. (B) Colocalization index of mRNA foci with SRSF2 speckles in each group shown in A. (C–E) Confocal microscopic images showing the JUND mRNA transcribed from the microinjected reporter construct and SRSF2 in HeLa cells treated with Cntl or MRT4/XRN2 siRNA for 72 h. The red and green lines in the graphs show the intensity of the FISH and SRSF2 IF signal, respectively (C). Colocalization indexes of the JUND FISH foci and SRSF2 dots at each time point are shown in D. Quantifications of nuclear and cytoplasmic JUND mRNA FISH signals are shown in E. Data represent the mean ± SD from three independent experiments; n = 10. Bars: 10 µm (magnification); 20 µm (others). Statistical analysis was performed using an unpaired t test. *, P < 0.05; ***, P < 0.01. KD, knockdown.

Figure 2.

Endogenous intronless mRNAs associate with NSs. (A) Confocal microscopic images to show distributions of endogenous (Endo) RNAs and NSs detected by FISH with transcript-specific probes and IF with the SRSF2 antibody. To clearly visualize the NEAT1 foci, a shorter exposure is shown. The green and red lines in the graphs demonstrate the RNA and SRSF2 IF signal intensity, respectively. The colocalization index of RNA foci with SRSF2 speckles is shown in the bottom. Data represent the mean ± SD from three independent experiments; n = 10. Statistical analysis was performed using an unpaired t test. ***, P < 0.01. (B) High magnification of a representative cell with the JUND mRNA and SRSF2 detected is shown. The further higher magnification of the boxed region is shown. (C–F) Same as in B except that the HSPA1A mRNA, RHOB mRNA, ZXDB mRNA, and NEAT1 lncRNA were detected. (G) The top to bottom sections of Z-stacked confocal images were taken in steps of 0.5 µm. Bars: 1 µm (magnifications); 20 µm (others).

Figure 3.

Endogenous intronless mRNAs are specifically detected in NSs. (A) FISH signals with a negative Cntl probe targeting the antisense transcript of the mouse long noncoding RNA Gm14635, SRSF2 IF, and DAPI staining are shown. (B) RT-PCRs to examine the knockdown efficiencies of endogenous intronless mRNAs in cells electronically transfected with ASOs for 48 h. (C–F) FISH signals of endogenous (Endo) intronless mRNAs in HeLa cells were microinjected with a Cntl ASO or ASO targeting the corresponding intronless mRNAs for 10 h. Note that the same exposures were taken for the cells treated with Cntl or transcript-specific ASO. (G and H) RT-qPCRs to examine the levels of the wG mRNA derived from microinjected constructs (G) or endogenous mRNAs (H) in HeLa cells mock-treated or treated with α-amanitin (4 µg/ml) for 6, 12, and 18 h. The graphs show the relative abundance of wG mRNA to DNA or relative abundance of COL1A1, PTB1, and DDX39B pre-mRNAs to 18S rRNA. Data represent the mean ± SEM; n = 3. (I–K) Confocal microscopic images show the distribution of endogenous HSPA1A (I) and ZXDB (J) mRNAs and NSs in HeLa cell mock-treated or treated with α-amanitin (4 µg/ml) for 18 h. The same exposures were taken for the cells treated with Cntl or α-amanitin. The average N/C ratios of each intronless mRNA are shown in the graph (K). The data represent the mean ± SD from three independent experiments; n = 10. Bars, 20 µm. The green and red lines in the graphs demonstrate the mRNA and SRSF2 IF signal intensity, respectively. Statistical analysis was performed using an unpaired t test. ***, P < 0.01.

Figure 4.

In vitro–transcribed naturally intronless mRNAs without a polyA tail are accumulated in NSs. (A) Top: Schematic of reporter constructs. Sequences from the vector and the genes of interest are indicated as cyan line and gray bar, respectively. The positions of the cap, polyA tail (An), and FISH probe are indicated. Confocal microscopic images show the distribution of microinjected cG, HSPA1A, GPR119, and JUND mRNAs as well as NSs. FISH with the 5′ vector probe and SRSF2 IF were performed 30 min after microinjection. Bar, 20 µm. The red and green lines in the graphs demonstrate the mRNA and SRSF2 IF signal intensity, respectively. The colocalization indexes are shown on the right. Data represent the mean ± SD from three independent experiments; n = 10. Statistical analysis was performed by using the unpaired Student’s t test. ***, P < 0.01. CDS, coding sequence. (B) Same as in A, except that in vitro–transcibed but not polyadenylated intronless mRNAs were used for microinjection.

Figure 5.

Multiple fragments in naturally intronless mRNAs facilitate speckle targeting. (A) Schematic of the HSPA1A constructs. Sequences from the vector and HSPA1A gene are indicated as a cyan line and an orange bar, respectively. The positions of the promoter, polyA (pA) signal, and FISH probe are marked. FL, full length. (B) Fluorescence microscopic images show the distribution of mRNA fragments transcribed from transfected DNA constructs shown in A. FISH with the 3′ vector probe was performed at 24 h after transfection. (C) Confocal microscopic images show the distribution of HSPA1A fragment mRNAs derived from microinjected DNA construct and NSs. FISH with the 3′ vector probe and SRSF2 IF were performed at 1 h after the addition of α-amanitin. The colocalization index is shown on the right. (D) Top: Schematic of cG constructs. Sequence from the vector is indicated as cyan line, and the regions of the cG and STEs are indicated as a gray and an orange bar, respectively. The positions of promoter, polyA site, and FISH probe are marked. Bottom: Fluorescence microscopic images show the distribution of cG, cG-STE1, and cG-STE2 mRNAs transcribed from microinjected DNA constructs and NSs. α-Amanitin (4 µg/ml) was added 20 min after microinjection. FISH with the 3′ probe and IF with the SRSF2 antibody were performed at 30 min after the addition of α-amanitin. The colocalization index is shown on the right. Data represent the mean ± SD from three independent experiments; n = 10. (E) Same as in D, except that the Smad cDNA transcript (cS) was used. Bars, 20 µm. The red and green lines in the graphs demonstrate the mRNA and SRSF2 IF signal intensities, respectively. Statistical analysis was performed by using the unpaired t test. ***, P < 0.01.

Figure 6.

ESEs and SR proteins function in speckle association of intronless mRNAs. (A) Top: Schematic of cG constructs. Sequence from the vector is indicated as the cyan line, and the regions of the cG and ESEs are indicated as gray and green bars, respectively. The positions of the promoter, polyA (pA) signal, and FISH probe are marked. Bottom: Fluorescence microscopic images show the distribution of cG, cG-ESE1, and cG-ESE2 mRNAs transcribed from microinjected DNA constructs and NSs. α-Amanitin (4 µg/ml) was added 20 min after microinjection. FISH with the 3′ probe and IF with the SRSF2 antibody were performed at 2 h after the addition of α-amanitin. The colocalization index is shown on the right. The red and green lines in the graphs demonstrate the mRNA and SRSF2 IF signal intensities, respectively. (B) Confocal fluorescence microscopic images to show the distribution of the cG-ESE2 mRNA coexpressed with the Cntl or ΔSR RNAs and NSs. α-Amanitin (4 µg/ml) was added 20 min after coinjection of the cG-ESE2 construct (30 ng/µl), together with the Cntl or the ΔSR construct (100 ng/µl). FISH with the β-globin probe and IF with the SRSF2 antibody were performed at 2 h after the addition of α-amanitin. The colocalization index is shown on the right. The green and red lines in the graphs demonstrate the mRNA and SRSF2 IF signal intensities, respectively. The colocalization index is shown on the right. (C and D) Confocal fluorescence microscopic images show the distribution of the endogenous HSPA1A (C) and RHOB (D) mRNAs and NSs in cells expressing the Cntl or ΔSR RNA. To allow the release of mRNAs that had entered NSs before SR depletion, 8 h after microinjection of the Cntl or ΔSR construct, FISH with transcript-specific probes, SRSF2 IF, and GFP IF indicating the microinjected cells (circles) were performed. Note that to avoid signal leakage between channels, only a tiny amount of GFP construct was injected. The green and red lines in the graphs demonstrate the mRNA and SRSF2 IF signal intensities, respectively. The colocalization index is shown at the bottom. (E) Confocal fluorescence microscopic images show the distribution of the endogenous HSPA1A and RHOB mRNAs and NSs in cells treated with Cntl or SRSF1/3/7 siRNAs for 72 h. The green and red lines in the graphs demonstrate the mRNA and PABPN1 IF signal intensities, respectively. The colocalization index is shown on the right. (F) Top: Schematic of cG and cG-M6 constructs. Sequences from the vector and cG are indicated as cyan line and gray bars, respectively. The positions of the promoter, polyA (pA) signal, and FISH probe are marked. Bottom: Confocal fluorescence microscopic images show the distribution of cG and cG-M6 transcribed from microinjected DNA constructs and NSs in cells coexpressing GFP-MS2 (Cntl) or MS2-SRSF2. α-Amanitin (4 µg/ml) was added 20 min after microinjection. FISH with the 3′ probe and IF with the PABPN1 antibody were performed at 2 h after the addition of α-amanitin. The colocalization index is shown on the right. The red and green lines in the graphs demonstrate the mRNA and PABPN1 IF signal intensities, respectively. Data represent the mean ± SD from three independent experiments; n = 10. Bars, 20 µm. Statistical analysis was performed using an unpaired t test. ***, P < 0.01.

Figure 7.

Speckle association promotes TREX recruitment and TREX-dependent intronless mRNA export. (A) Fluorescence microscopic images show the distribution of the cG, cG-ESE1, and cG-ESE2 mRNA derived from equal amounts of microinjected cG, cG-ESE1, and cG-ESE2 constructs. α-Amanitin (4 µg/ml) was added at 20 min after injection, and 4 h later, FISH with the 3′ vector probe was performed. DAPI staining marks the nucleus. Relative N/C ratios are shown on the right. Data represent the mean ± SD from three independent experiments; n = 30. (B) Equal amounts of cG, cG-ESE1, and cG-ESE2 constructs were transfected into HeLa cells. 24 h later, N/C fractionation was performed. Western blotting (WB) was used to examine the purities of the nuclear and cytoplasmic fraction by using UAP56 and tubulin as markers (left). Ratios of nuclear to cytoplasmic β-globin mRNAs after normalization to NEAT1 (nucleus) and GAPDH (cytoplasm) in each fraction are presented in the bar graph (right). Data represent the mean ± SEM; n = 3. (C) Western analysis was used to examine the knockdown (KD) efficiency of UAP56. (D) Similar to A, except that Cntl or UAP56 knockdown cells were used. Note that to achieve efficient mRNA nuclear retention and to avoid secondary effect, the cells were treated with UAP56 and URH49 siRNAs for 48 h. Data represent the mean ± SD from three independent experiments; n = 30. (E) RIPs with UV cross-linking were performed with an ALYREF antibody or IgG (see details in Materials and methods). Western analysis was performed by using the ALYREF antibody (left). The relative IP efficiencies of cG mRNAs to the cS mRNA were quantified and are indicated in the graph (right). Data represent the mean ± SEM; n = 3. (F) Fluorescence microscopic images show the distribution of the cG-ESE2 mRNA derived from the microinjected cG-ESE2 construct. α-Amanitin (4 µg/ml) was added at 20 min after injection, and 4 h later, FISH with the β-globin probe was performed. DAPI staining marks the nucleus. Relative N/C ratios are shown on the right. Data represent the mean ± SD from three independent experiments; n = 30. (G) The graph shows the average N/C ratios of the endogenous HSPA1A and RHOB mRNAs in cells shown in Fig. 6 E. Data represent the mean ± SD from three independent experiments; n = 30. (H) Fluorescence microscopic images show the distribution of the cG or cG-M6 mRNAs derived from microinjected constructs in cells coexpressing GFP-MS2 or MS2-SRSF2. FISH with the 3′ vector probe was performed at 4 h after injection. The graph shows the relative N/C ratios. Data represent the mean ± SD from three independent experiments; n = 30. (I) Similar to H, except that the cG-M6 and MS2-SRSF2 were coinjected into Cntl or UAP56 knockdown cells (48 h knockdown). Data represent the mean ± SD from three independent experiments; n = 30. Bars, 20 µm. Statistical analysis was performed using an unpaired t test. ***, P < 0.01. Molecular masses are given in kilodaltons.

Figure 8.

NXF1 depletion results in nuclear mRNA retention both inside and outside of NSs. (A and B) Confocal microscopic images show the JUND mRNA transcribed from the microinjected reporter construct and SRSF2 in HeLa cells treated with UAP56/URH49 siRNA for 48 h. The red and green lines in the graphs show the intensity of the FISH and SRSF2 IF signal, respectively (A). Colocalization indexes of the JUND FISH foci and SRSF2 dots (left) and total FISH signal quantification (right) at each time point are shown in B. (C) Western analysis was used to examine the knockdown (KD) efficiency of NXF1. (D) Confocal microscopic images show the endogenous RHOB mRNA and NSs (SRSF2) in HeLa cells treated with Cntl, UAP56/URH49, or NXF1siRNAs for 48 h. Higher magnification of the boxed regions is shown. The green and red in the line scan graphs demonstrate the intensities of the mRNA and the SRSF2 IF signals, respectively. The bar graphs show the relative N/C ratios (top) and colocalization index (bottom) in each treatment group. Data represent the mean ± SD from three independent experiments; n = 10. (E) Same as in D, except that the endogenous HSPA1A mRNA was examined. (F) Same as in D, except that the bulk polyA RNAs were examined. Bars, 20 µm. Statistical analysis was performed by using the unpaired Student’s t test. ***, P < 0.01. Molecular masses are given in kilodaltons.

Figure 9.

Nucleoplasmic accumulated mRNAs upon NXF1 depletion had passed through NSs and recruited export factors. (A) Western analysis was used to examine the knockdown (KD) efficiency of NXF1 and UAP56. Note that to achieve efficient mRNA nuclear retention and to avoid extensive cell death, the cells were treated with UAP56 and URH49 siRNAs for 36 h. (B and C) Confocal microscopic images show the distribution of polyA RNAs (B) or the endogenous HSPA1A mRNA (C) and NSs (SRSF2) in HeLa cells treated with Cntl or UAP56/URH49/NXF1 siRNAs for 36 h. The green and red lines in the graphs demonstrate the RNA and SRSF2 IF signal intensities, respectively. Colocalization indexes are shown on the right. (D and E) Confocal microscopic images show the distribution of polyA RNAs (D) or the endogenous (Endo.) HSPA1A mRNA (E), UAP56, and NSs (SRSF2) in HeLa cells treated with Cntl or NXF1 siRNA for 48 h. The green, red, and blue lines in the graphs demonstrate the RNA, UAP56, and SRSF2 signal intensities, respectively. Colocalization indexes are shown on the right. Data represent the mean ± SD from three independent experiments; n = 10. (F) mRNAs derived from intron-containing genes associate with NSs dependent on splicing, whereas naturally intronless mRNAs enter these subnuclear structures via SR proteins that are bound on ESEs. Aberrant mRNAs such as cDNA transcripts cannot enter NS due to lack of splicing. In NSs, mRNA export factors are efficiently recruited due to their high concentration, resulting in efficient nuclear export of spliced and intronless mRNAs. Aberrant mRNAs have a limited chance to get access to mRNA export factors and are mostly retained in the nucleus. Bars, 20 µm. Statistical analysis was performed using an unpaired t test. ***, P < 0.01. Molecular masses are given in kilodaltons.