mRNAs are sorted for export or degradation before passing through nuclear speckles

Abstract

A significant fraction of mRNAs are degraded by the nuclear exosome in normal cells. Here, we studied where and when these exosome target mRNAs are sorted away from properly exported ones in the cells. We show that upon exosome inactivation, polyA RNAs are apparently accumulated in nuclear foci that are distinct from nuclear speckles (NSs), and provide several lines of evidence supporting that these polyA RNAs mainly correspond to accumulating exosome target mRNAs. These results suggest that exosomal mRNA degradation mostly occurs outside of NSs. In support of this possibility, targeting exosome target mRNAs to NSs stabilizes them by preventing exosomal degradation. Furthermore, inhibiting mRNA release from NSs does not attenuate exosomal degradation in normal cells, and results in polyA RNA accumulation both inside and outside of NSs in exosome inactivated cells, suggesting that passage through NSs is not required for sorting mRNAs for degradation or export. Indeed, exosome target mRNAs that normally do not enter NSs are exported upon exosome inactivation. Together, our data suggest that exosome target mRNAs are mainly degraded in the nucleoplasm before entering NSs and rapid removal of these mRNAs is important for preventing their nuclear export.

Figure 1.

Exosome inactivation results in the accumulation of polyA RNAs in specific nuclear foci. (A) Western and RT-PCR data showing knockdown efficiencies of the exosome components and MTR4. Different amount of cells or RT products of control knockdown cells were used to estimate the knockdown efficiencies. (B) FISH analysis of polyA RNA distribution in exosome and MTR4 knockdown cells. Same exposure was taken for all images. DAPI staining served as nucleus marker. (C) Quantification of nuclear polyA RNA FISH signals. Nuclear polyA RNA FISH signals quantified from 30 cells in each experiment by Image J. Error bars represent standard deviations from biological repeats (n = 3). Statistical analysis was performed using Student’s t-test. *P < 0.05, **P < 0.01. (D) Wild-type, but not helicase core mutant MTR4 repressed the nuclear polyA RNA accumulation phenotype in MTR4 knockdown cells. Domain schematic representation of MTR4 is shown on the top. Functional domains are indicated and point mutations of D252A and E253A are marked. The MTR4 siRNA was transfected into HeLa cells using Lipofectamine 2000. Forty-eight hours post-transfection, siRNA sensitive or resistant WT Flag-MTR4, or siRNA resistant helicase core mutant MTR4 expression plasmid, was transfected to MTR4 siRNA treated cells. Twenty-four hours post-transfection, FISH analysis was carried out to observe the distribution of polyA RNAs. IF with the Flag antibody was performed to examine exogenous MTR4 expression. DAPI staining served as nucleus marker. The arrows indicate cells for which nuclear polyA RNA accumulation phenotype was repressed by exogenously expressed MTR4. (E) Nuclear accumulated polyA RNAs do not co-localize with NSs, paraspeckles, Cajal bodies or PML bodies in RRP40 knockdown cells. FISH was carried out using the 70 (nt) oligo-dT probe and IF using indicated antibodies were carried out. DAPI staining served as the nucleus marker. Confocal microscopy was used to visualize the cells.

Figure 2.

mRNA nuclear export is not apparently affected by exosome inactivation. (A) Western blots to examine the knockdown efficiency of UAP56. GAPDH is used as a loading control. (B) Nuclear export of the HSPA1A reporter mRNA is not affected in exosome and MTR4 knockdown cells. The HSPA1A reporter mRNA construct was injected into the nuclei of control, UAP56, RRP40 and MTR4 knockdown cells, followed by FISH to detect the distribution of HSPA1A mRNA at 4 h after injection. Inset images show the injection marker. Same exposure was taken for all images. Quantification of nuclear and cytoplasmic FISH signals of the HSPA1A mRNA. N/C ratios were determined for 30 cells in each experiment. C and N indicate cytoplasmic and nuclear FISH signals, respectively. Error bars, standard deviations (n = 3). Statistical analysis was performed using Student’s t-test. ***P < 0.001. (C) PolyA RNA distribution in UAP56, RRP6/DIS3 and MTR4 knockdown cells. Indicated siRNAs were transfected into HeLa cells. Seventy-two hours post-transfection, FISH was carried out to observe the distribution of polyA RNAs. DAPI staining served as nucleus marker. Note that to accurately quantify the polyA signals in all samples, same exposures were taken for all FISH images. (D) Quantification of nuclear and cytoplasmic polyA RNA FISH signals. Nuclear and cytoplasmic polyA RNA FISH signals quantified from 50 cells in each experiment by Image J. Error bars represent standard deviations from biological repeats (n = 3). Statistical analysis was performed using Student’s t-test. *P < 0.05, **P < 0.01.

Figure 3.

Increased nuclear polyA signals upon exosome inactivation is not mainly due to the lengthened polyA tail. (A) Western blotting to examine the knockdown efficiencies of MTR4 and PABPN1. HeLa cells were transfected with siRNAs targeting the indicated genes. Seventy-two hours post-transfection, western blotting was carried out with indicated antibodies. GAPDH is used as a loading control. (B) Western blots to examine the purities of nuclear and cytoplasmic fractions prepared from control, MTR4, PABPN1 and MTR4/PABPN1 knockdown cells. UAP56 and tubulin are used as nuclear and cytoplasmic marker, respectively. (C) Nuclear polyA RNA tail analysis from cells transfected with indicated siRNAs. (D) PolyA RNA distribution in MTR4, MTR4/PABPN1 knockdown cells. HeLa cells transfected with indicated siRNAs were used for FISH analysis to examine the distribution of polyA RNAs. DAPI staining was used to indicate the nuclei. Note that to accurately quantify the polyA signals in all samples, same exposures were taken for all FISH images. (E) Quantification of total polyA RNA FISH signals. Total polyA RNA FISH signals quantified from 30 cells in each experiment by Image J. Error bar represent standard deviations from three biological replicates. Statistical analysis was performed using Student’s t-test. ***P < 0.001. (F) Nuclear RNA-seq signal shows that the spliced RHOC mRNA is accumulated in RRP40 knockdown cells. (G) (Left) FISH signals of the endogenous RHOC mRNA in control and RRP40 knockdown cells. (Right) Quantification of nuclear RHOC mRNA FISH signals. Nuclear RHOC mRNA FISH signals were quantified from 30 cells in three experiment by Image J. Error bar represent standard deviations from biological repeats (n = 3). Statistical analysis was performed using Student’s t-test. ***P < 0.001.

Figure 4.

Nuclear polyA foci might be mainly formed by exosome target mRNAs. (A) The pie chart represents quantitative distribution of the RNAs whose RPM were elevated more than 1.5-fold in MTR4 knockdown relative to control knockdown. Each category represents RNAs unique to that category and non-overlapping with previous categories, with the initial category designated as ‘short ncRNA’ and proceeding clockwise. (B) Same as (A), except that the read distributions of the accumulated RNAs were shown. (C) Boxplots represent the distribution of nuclear accumulated reads along different parts of the mRNA in MTR4 knockdown cells at the genome-wide scale. Each mRNA is divided into 5′, middle, 3′ parts with the equal length. (D) Localization of the RHOC mRNA in control and RRP40 knockdown cells. SC35 and DAPI staining served as a marker for NS and nucleus, respectively. Confocal microscopy was used to visualize the cells. Arrowheads indicate RHOC mRNA that co-localized with polyA RNAs. Examples of polyA RNA foci that do not apparently co-localize with the RHOC mRNA are indicated by arrows. (E) Quantification of punctate FISH signal of the RHOC mRNA that localized in nuclear foci formed by polyA RNAs. The bars in the graph indicate the percentage of polyA-positive RHOC punctate FISH signal. (F and G) Same as (D and E), except that the DDX39B mRNA was detected in control and MTR4 knockdown cells.

Figure 5.

Formation of polyA foci upon exosome inactivation is not mainly due to accumulation of TRAMP, NEXT or ZFC3H1 substrates. (A) Western blotting show that NEXT components were efficiently knocked down. Tubulin is used as a loading control. (B) PROMPTs accumulate similarly in RRP40 and NEXT knockdown cells. HeLa cells were transfected with control, RRP40, RBM7, ZCCHC8 and RBM7/ZCCHC8 siRNA, 72 h post-transfection, polyA RNAs were prepared followed by RT-qPCRs with primer sets that specifically amplify the indicated PROMPTs. R7 and Z8 represent the RBM7and ZCCHC8, respectively. The bars show RNA levels relative to GAPDH. Error bars represent standard deviations from biological repeats (n = 3). Statistical analysis was performed using Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001. (C) PolyA RNA distribution is not affected in NEXT knockdown cells. HeLa cells transfected with indicated siRNAs were used for FISH analysis to observe the distribution of polyA RNAs. DAPI staining served as nucleus marker. (D) Quantification of nuclear polyA RNA FISH signals from 30 cells in each experiment by Image J. Error bar represent standard deviations from three biological replicates. ***P < 0.001. (E) The relative read abundance of PROMPTs and mRNAs in MTR4 knockdown cell. (F) Western blotting results show that TRAMP components were efficiently knocked down. Tubulin is used as a loading control. (G) Same as (C), except that TRAMP knockdown cells were used for this experiment. (H) Quantification of nuclear polyA RNA FISH signals from 30 cells in each experiment by Image J. Error bar represent standard deviations from three biological replicates. Statistical analysis was performed using Student’s t-test. ***P < 0.001. (I) RT-qPCR to examine the knockdown efficiency of ZFC3H1. Error bars represent standard deviations from biological repeats (n = 3). Statistical analysis was performed using Student’s t-test. ***P < 0.001. (J) RT-qPCR to examine SNHG transcript levels in control, MTR4 and ZFC3H1 knockdown cells. The bars show RNA levels relative to 18S rRNA. Error bars represent standard deviations from biological repeats (n = 3). Statistical analysis was performed using Student’s t-test. **P < 0.01, ***P < 0.001. (K) FISH analysis of polyA RNA distribution in ZFC3H1 knockdown cells. (Left panel) HeLa cells transfected with ZFC3H1 siRNAs were used for FISH analysis to observe the distribution of polyA RNAs. DAPI staining served as nucleus marker. (Right panel) Quantification of nuclear polyA RNA FISH signals from 30 cells in each experiment by Image J. Error bar represent standard deviations from three biological replicates. ***P < 0.001. (L) Nuclear accumulated polyA RNAs in ZFC3H1 knockdown cells co-localize with NSs, FISH was carried out using the 70 (nt) oligo-dT probe and IF using the SC35 antibody were carried out. Confocal microscopy was used to visualize the cells.

Figure 6.

Exosome target mRNAs are mainly degraded before passing through NSs. (A) Speckle targeting of the cG mRNA prevents exosome degradation. RT-qPCRs to examine the levels of cG mRNA or cG -STE mRNAs in normal HeLa cells (left panel), or in control and MTR4 knockdown cells (right panel). (B) Knockdown of PABPN1 and UAP56 inhibit mRNA release from NSs. Confocal microscopy was used to examine distribution of polyA RNAs in control, PABPN1 and UAP56 knockdown cells. SC35 was used to mark NSs. (C) RT-qPCRs to examine levels of indicated mRNAs from HeLa cells treated with control, MTR4, PABPN1 or UAP56/URH49 siRNAs. The relative levels of indicated RNAs to 18S rRNA were quantified and indicated in the graph. Error bars represent standard deviations from biological repeats (n = 3). Statistical analysis was performed using Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001. (D) Two possibilities in accumulation sites of exosome target mRNAs when mRNA release from NSs is inhibited. (I) They would accumulate in both inside and outside of NSs if the degradation occurs before entering NSs; (II) they would accumulate in exclusively inside of NSs if the degradation occurs after being released from NSs. (E) (Top) Confocal microscopy was used to examine the co-localization of polyA RNAs with NSs in control, MTR4, MTR4/PABPN1 and MTR4/UAP56 knockdown cells. (Bottom) Quantification of polyA foci that do not co-localize with SC35 in each cell. Thirty cells were used for the analysis for each sample. (F) MTR4 depletion results in cytoplasmic accumulation of the cG and cS transcripts. (Top) The cG or cS reporter construct was injected into the nuclei of control and MTR4 knockdown cells, followed by FISH using vector probe to detect the distribution of corresponding mRNA at 2 h later after injection. Same exposure was taken for all images. Inset images show the injection marker. DAPI staining served as nucleus marker. (Bottom) Quantification of nuclear and cytoplasmic FISH signals of the corresponding mRNA. N/C ratios were determined for 30 cells in each experiment. N and C indicate nuclear and cytoplasmic FISH signals, respectively. Error bars, standard deviations (n = 3). ***P < 0.001. (G) (Top) The cS reporter construct was injected into the nuclei of MTR4 and MTR/UAP56 knockdown cells, followed by FISH to detect the distribution of corresponding mRNA at 2 h post-injection. Same exposure was taken for all images. Inset images show the injection marker. DAPI staining served as nucleus marker. (Bottom) Quantification of nuclear and cytoplasmic FISH signals of the cS mRNA. N/C ratios were determined for 30 cells in each experiment. N and C indicate nuclear and cytoplasmic FISH signals, respectively. Error bars, standard deviations (n = 3). ***P < 0.001. (H) Nuclear accumulated cS mRNA in MTR4/UAP56 knockdown cells partially co-localize with NSs. FISH with vector probe and IF using the SC35 antibody were carried out.

Figure 7.

mRNA fate determination for export and degradation mainly occurs before mRNAs enter NSs. Left, in normal cells, on the mRNA, ALYREF and MTR4 competes for binding with CBC. If ALYREF outcompetes MTR4, the mRNA then enters NSs, where it might further recruit ALYREF via other mechanisms as well as other TREX components. Following its release from NSs, the mRNA is exported to the cytoplasm. In the case that MTR4 outcompetes ALYREF, the mRNA is then degraded in the nucleoplasm. Right, in exosome inactivated cells, exosome target mRNAs are mostly detected in the nucleoplasm due to their inefficient recruitment of ALYREF. A part of these stabilized target mRNAs are gradually exported to the cytoplasm through or not through NSs.