Exosome cofactor hMTR4 competes with export adaptor ALYREF to ensure balanced nuclear RNA pools for degradation and export

Abstract

The exosome is a key RNA machine that functions in the degradation of unwanted RNAs. Here, we found that significant fractions of precursors and mature forms of mRNAs and long noncoding RNAs are degraded by the nuclear exosome in normal human cells. Exosome-mediated degradation of these RNAs requires its cofactor hMTR4. Significantly, hMTR4 plays a key role in specifically recruiting the exosome to its targets. Furthermore, we provide several lines of evidence indicating that hMTR4 executes this role by directly competing with the mRNA export adaptor ALYREF for associating with ARS2, a component of the cap-binding complex (CBC), and this competition is critical for determining whether an RNA is degraded or exported to the cytoplasm. Together, our results indicate that the competition between hMTR4 and ALYREF determines exosome recruitment and functions in creating balanced nuclear RNA pools for degradation and export.

Keywords: ALYREF; ARS2; hMTR4; mRNA export; nuclear RNA degradation.

Figure 1. Genome‐wide study of in vivo targets of the human nuclear exosome

1. A

   A diagram of the RNA‐seq experimental approach.

2. B

   Western blotting to examine the knockdown efficiencies of hRRP40 and hMTR4. Tubulin was used as a loading control. Different amounts of control knockdown samples were loaded to estimate the knockdown efficiencies.

3. C

   Western blotting to examine the purity of nuclear fractions. Nuclear proteins UAP56, hMTR4, and hRRP6 and the cytoplasmic protein tubulin served as the nuclear and cytoplasmic markers, respectively. N, nucleus; C, cytoplasm. The asterisk indicates a nonspecific band that is detected by the hRRP6 antibody.

4. D

   The distribution of reads derived from RNA‐seq libraries and mapped to the indicated RNA classes. Each category represents RNAs unique to that category and nonoverlapping with previous categories. In hRRP40 knockdown cells, Fisher's exact test, lncRNA, adjusted P = 0, odds ratio = 1.10; repetitive elements, adjusted P = 0, odds ratio = 1.47; and others, adjusted P = 0, odds ratio = 1.37. In hMTR4 knockdown cells, Fisher's exact test, lncRNA, adjusted P = 0, odds ratio = 1.18; repetitive elements, adjusted P = 0, odds ratio = 1.75; others, adjusted P = 0, odds ratio = 2.06; and short ncRNA, adjusted P = 0, odds ratio = 1.53.

5. E

   The Venn diagrams depict the overlap of accumulated snRNAs in hRRP40 and hMTR4 knockdown cells. The numbers in Venn diagrams show snRNAs whose RPM were elevated more than 1.5‐fold in hRRP40 or hMTR4 knockdown relative to control knockdown cells. Note that only sequencing reads mapping to the gene bodies were computed. The percentages of snRNAs that increased more than 1.5‐fold in hRRP40 and hMTR4 knockdown are shown. Statistical analysis P‐value was used to measure the overlapping of genes regulated by hRRP40 and hMTR4 and performed using Fisher's exact test by R language.

6. F–L

   Same as (E), except that instead of snRNAs, accumulated snoRNAs, PROMPTs, eRNAs, intron‐containing mRNAs, intronless mRNAs, intron‐containing lncRNAs, and intronless lncRNAs are shown, respectively. Note that relative to mRNAs, more lncRNAs were accumulated in both hRRP40 (26% for mRNAs and 50% for lncRNAs according to exon changes) and hMTR4 (26% for mRNAs and 57% for lncRNAs according to exon changes) knockdown cells. In hMTR4 knockdown, compared to intron‐containing mRNAs, relatively more intronless mRNAs were accumulated (according to exon reads change, compare 43 to 26%, Fisher's exact test P < 0.0001, odds ratio = 1.67).

Source data are available online for this figure.

Figure 2. Five categories of mRNAs based on level change upon exosome knockdown

1. The levels of both precursor and mature forms are elevated. (Top) RNA‐seq signal of TCTA is shown as an example. The blue and purple lines mark the position of PCR products for pre‐mRNAs and spliced mRNAs, respectively. The intron regions are boxed in color. Numbers to the left show the RPM. (Bottom) RT–qPCRs to specifically amplify pre‐mRNAs and spliced mRNAs in the nuclei of control, hRRP40, and hMTR4 knockdown cells. The bars show RNA levels relative to 18S rRNA. Error bars, standard deviations (n = 3). Statistical analysis was performed using Student's t‐test. *P < 0.05, **P < 0.01.

2. Same as (A), except that in this category, the levels of pre‐mRNAs increase with no apparent change in those of spliced mRNAs.

3. Same as (A), except that in this category, the levels of spliced mRNAs increase with no apparent change in those of pre‐mRNAs.

4. Same as (A), except that in this category, the levels of intronless mRNAs significantly increase.

5. Same as (A), except that in this category, the levels of neither precursors nor mature forms significantly increase.

6. The distribution of exosome target mRNAs and lncRNAs falling in each category.

Figure EV1. Deep‐sequencing signals of mRNAs that were examined in Fig 2

1. Deep‐sequencing signals of exosome targets in each category are shown. (I) The levels of both precursor and mature forms are elevated. (II) The levels of pre‐mRNAs increase with no apparent change in those of spliced mRNAs. (III) The levels of spliced mRNAs increase with no apparent change in those of pre‐mRNAs. (IV) The levels of intronless mRNAs significantly increase. (V) The levels of neither precursors nor mature forms significantly increase.

2. Illustration of the position of RT–qPCR products on the indicated gene. The blue and purple lines show the PCR product of pre‐mRNAs and spliced mRNAs, respectively.

Figure EV2. Full length of the DNAJC30 and NDUFAF3 mRNAs might be stabilized in hRRP40 or hMTR4 knockdown cells

(Top) Deep‐sequencing signals of DNAJC30 and NDUFAF3 are shown. The plot of RNA‐seq data for DNAJC30 is the same as that in Fig 2D. The blue lines mark the position of qPCR products. (Bottom) RT–qPCRs to examine the nuclear levels of different parts of the mRNAs relative to the 18S rRNA in control, hRRP40, and hMTR4 knockdown cells. Statistical analysis was performed using Student's t‐test. Error bars represent standard deviations from biological repeats (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure EV3. Transcription elevation does not account for the increased levels of most exosome targets

1. The pie chart represents populations of genes for which the RNAPII binding was enhanced, remained unchanged, or was reduced.

2. Examples of RNAPII ChIP‐seq data for genes belonging to different categories.

3. The distribution of genes with reduced RNAPII binding in hRRP40 knockdown cells falling in each category according to their level change.

Figure 3. Identification of nuclear accumulated mRNAs and lncRNAs in hMTR4 knockdown cells

1. A

   A diagram of the RNA‐seq experimental approach.

2. B

   Western blotting to examine hMTR4 knockdown efficiency and purities of nuclear fractions. UAP56 and tubulin were used as nuclear and cytoplasmic markers, respectively. N, nucleus; C, cytoplasm.

3. C

   The distribution of reads derived from RNA‐seq libraries and mapped to the indicated RNA classes. Each category represents RNAs unique to that category and nonoverlapping with previous categories. Calculated mean of mapped reads in triplicate data, Fisher's exact test, lncRNAs, adjusted P = 0, odds ratio = 1.15; repetitive elements, adjusted P = 0, odds ratio = 1.66; and others, adjusted P = 0, odds ratio = 1.42.

4. D

   The Venn diagrams depict the overlap of accumulated RNAs (> 1.5‐fold) detected by nuclear total RNA‐seq with those detected by nuclear polyA RNA‐seq in hMTR4 knockdown cells. Fisher's exact test P < 0.0001, odds ratio = 2.90.

5. E

   The Venn diagrams depict the accumulated intron‐containing mRNAs in hMTR4 knockdown cells. The numbers in Venn diagrams show exon or intron reads that are elevated more than 1.5‐fold in hMTR4 knockdown cells. The percentages of intron‐containing mRNAs accumulated more than 1.5‐fold in hMTR4 knockdown according to exon or intron changes are shown.

6. F–H

   Same as (E), except that instead of accumulated intron‐containing mRNAs, intronless mRNAs, intron‐containing lncRNAs, and intronless lncRNAs are shown, respectively. Note that relatively more lncRNAs are accumulated than mRNAs in hMTR4 knockdown cells (Fisher's exact test P < 0.0001, odds ratio = 3.46 according to exon change).

7. I

   Box and whisker plots showing hMTR4 sensitivity (log2 (hMTR4 siRNA knockdown RPM/control RPM)) of pre‐mRNAs, spliced mRNAs, and intronless mRNAs. Note that this analysis was done to the all detected genes in RNA‐seq. Plot‐whisker: min to max. The difference between spliced mRNA and intronless mRNA is statistically significant (Wilcoxon test. ***P < 0.001).

8. J

   Same as (I), except that pre‐lncRNAs, spliced lncRNAs and intronless lncRNAs are shown. The difference between spliced and intronless lncRNAs is statistically insignificant (Wilcoxon test. P = 0.38).

Source data are available online for this figure.

Figure 4. hMTR4 functions in recruiting the exosome to its targets

1. Export‐defective β‐globin cDNA transcript is a nuclear exosome target. RT–qPCRs to examine the level of β‐globin cDNA transcript in control, hRRP40, and hMTR4 knockdown cells. The relative level of β‐globin cDNA transcript to the transfection control HSPA1A, which is not an exosome target, is quantified and indicated in the graph.

2. Export‐proficient β‐globin spliced mRNA is not a nuclear exosome target. Same as (A), except that β‐globin pre‐mRNA reporter construct was used.

3. Export‐defective Smad cDNA transcript is a nuclear exosome target. Same as (A), except that the Smad cDNA reporter construct was used.

4. Export‐proficient Smad spliced mRNA is not a nuclear exosome target. Same as (A), except that Smad pre‐mRNA reporter construct was used.

5. hMTR4 preferentially associates with the β‐globin cDNA transcript. β‐globin spliced or cDNA reporter construct was transfected into Flag‐hMTR4 stable expression cells. 6 h post‐transfection, RNAs immunoprecipitated by ALYREF, Flag, and Myc antibodies were used for RT–qPCR analysis. The relative level of the β‐globin mRNA to GAPDH was quantified and indicated in the graph. Note that primers specifically amplifying the β‐globin spliced mRNA were used for PCRs.

6. hMTR3 preferentially associates with the β‐globin cDNA transcript. Same as (E), except that Flag‐hMTR3 stable expression cells were used and IPs were carried out using the Flag and the Myc antibodies.

7. hMTR4 is required for efficient association of the exosome with its targets. β‐globin cDNA reporter construct was co‐transfected with tRNA construct into control or hMTR4 siRNA‐treated Flag‐hMTR3 stable expression cells. The relative level of β‐globin cDNA transcript to tRNA present in the immunoprecipitate was quantified and indicated in the graph.

8. The exosome is not required for efficient association of hMTR4 with exosome targets. Same as (G), except that control or hRRP40 siRNA‐treated Flag‐hMTR4 stable expression cells were used.

Data information: (A–H) 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. Source data are available online for this figure.

Figure EV4. TRAMP or NEXT might not be generally involved in the degradation of most mRNAs and lncRNAs

1. Western blotting results show that TRAMP and NEXT components were efficiently knocked down.

2. Western blotting to estimate knockdown efficiencies of TRAMP components. HeLa cells expressing Flag‐ZCCHC7 were transfected with ZCCHC7, PAPD5, or control siRNA. 72 h post‐transfection, Western blotting was carried out to examine the exogenously expressed Flag‐ZCCHC7 or the endogenous PAPD5. Tubulin is used as a loading control. Different amounts of cell lysates of control knockdown cells were loaded to estimate the knockdown efficiencies.

3. Same as (B), except that Flag‐RBM7 expression plasmid was transfected to NEXT component knockdown cells.

4. β‐globin cDNA reporter together with the HSPA1A control plasmid was transfected into control‐, hMTR4‐, PAPD5‐, ZCCHC7‐, RBM7‐, or ZCCHC8 siRNA‐treated HeLa cells. 12 h post‐transfection, total RNAs were extracted followed by RT–qPCRs. The graph shows relative level of β‐globin mRNA to the HSPA1A mRNA. Error bars represent standard deviations from biological repeats (n = 3). Statistical analysis was performed using Student's t‐test. **P < 0.01.

5. RT–qPCRs to examine the levels of indicated mRNAs in HeLa cells treated with control, hMTR4, PAPD5, ZCCHC7, and PAPD5/ZCCHC7 siRNA. The relative levels of indicated RNAs to 18S rRNA are shown in the graph. Statistical analysis was performed using Student's t‐test. Error bars represent standard deviations from biological repeats (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.

6. Same as (E), except that HeLa cells were treated with control, hMTR4, RBM7, ZCCHC8, or RBM7/ZCCHC8 siRNA.

Source data are available online for this figure.

Figure 5. Both hMTR4 and ALYREF interact with ARS2 in vivo and in vitro

1. The list of hMTR4‐associating proteins. IPs with the Flag or a control antibody were carried out from the RNased nuclear extract of the Flag‐hMTR4 stable expression cells. Proteins specifically identified in the Flag immunoprecipitate by mass spectrometry are listed. Unique, matched queries; total, matched peptides; MW, molecular weight. Note that ARS2 is detected in the Flag immunoprecipitate.

2. The list of ALYREF‐associating proteins. IPs with the ALYREF or a control antibody were carried out from RNased HeLa nuclear extract. Proteins specifically detected in the ALYREF immunoprecipitate by mass spectrometry are listed. Note that ARS2 was detected in the ALYREF immunoprecipitate.

3. (Left) ARS2 interacts with hMTR4 in vivo. IPs with the ARS2 or the hMTR4 antibody, or IgG were carried out from the RNase‐treated HeLa nuclear extract followed by Western blotting. 3% of the inputs were loaded. (Middle) ARS2 and ALYREF interact in vivo. IPs with the ARS2, ALYREF, and Myc antibody were carried out from the RNase‐treated HeLa nuclear extract followed by Western blotting. 10% of the inputs were loaded. (Right) hMTR4 and ALYREF have no interaction in vivo. IPs with the ALYREF, hMTR4, and IgG antibody were carried out from the RNase‐treated HeLa nuclear extract followed by Western blotting. 3% of the inputs were loaded.

4. ALYREF directly interacts with ARS2 in vitro. Purified GST and GST‐ARS2 proteins were used for pulling down purified MBP‐ALYREF or MBP in the presence of RNase A. Proteins pulled down were separated by SDS–PAGE followed by Coomassie staining. 37.5% of the inputs were loaded.

5. hMTR4 interacts with ARS2 in vitro directly. GST‐ARS2 and the negative control GST‐eIF4A3 were used for pull‐down of purified MBP‐hMTR4 or MBP in the presence of RNase A. Proteins pulled down were separated by SDS–PAGE, followed by Coomassie staining and Western blotting. 37.5% of the inputs were loaded.

Source data are available online for this figure.

Figure 6. hMTR4 competes with ALYREF for associating with ARS2 and RNAs

1. A

   hMTR4 competes with ALYREF for associating with ARS2 in vivo. HeLa cells overexpressing Flag‐DDX3 (control) or Flag‐ALYREF were used for IPs in the presence of RNase A with the ARS2, hMTR4, or Myc antibody, followed by Western blotting. 3% of the inputs were loaded.

2. B

   Same as (A), except that instead of the ARS2 antibody, the CBP80 antibody was used.

3. C

   hMTR4 competes with ALYREF for interacting with ARS2 in vitro. Pull‐down of MBP‐hMTR4 with GST‐ARS2 was carried out in the presence of MBP or MBP‐ALYREF under RNase‐treated condition.

4. D, E

   RNA‐IP analysis showed that the association of hMTR4 with RNAs was weakened by ALYREF overexpression. IPs were carried out with the hMTR4 antibody from Flag‐DDX3 (control) or Flag‐ALYREF‐overexpressing HeLa cells. The immunoprecipitates were used for RT–qPCRs to detect immunoprecipitated RNAs (D) and Western analysis to detect immunoprecipitated hMTR4 (E). Statistical analysis was performed using Student's t‐test. Error bars, standard deviations (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.

5. F

   Exosome target RNAs were accumulated in ALYREF‐overexpressing cells. Flag‐DDX3 or Flag‐ALYREF expression plasmid was transfected into HeLa cells followed by RT–qPCRs to detect levels of indicated RNAs. The relative levels of indicated RNAs to 18S rRNA were quantified and indicated in the graph. Statistical analysis was performed using Student's t‐test. Error bars, standard deviations (n = 3). *P < 0.05.

6. G, H

   Genome‐wide effect of ALYREF overexpression on the association of hMTR4 with RNAs. IPs with the hMTR4 antibody were carried out from control or Flag‐ALYREF overexpression HeLa cells. The immunoprecipitates were used for Western analysis (G) and deep sequencing (H). The experiment was biologically repeated for three times. (G) Western blotting with the hMTR4 antibody to detect the IP efficiencies. (H) (Left graph) According to the change in hMTR4 association upon ALYREF overexpression, the RNAs are grouped into four classes. The percentages of RNAs falling in each class are shown. (Right graph) RNAs in the < 0.5 class were further grouped into three classes according to their level change detected by nuclear total RNA‐seq shown in Fig 1.

7. I

   Screenshots of two replicates of hMTR4 RIP‐seq signals of DNAJC30 and H‐AS1 are shown.

8. J

   Western blotting to examine the purities of nuclear fractions prepared from control and ALYREF overexpression cells. UAP56 and tubulin serve as the nuclear and cytoplasmic markers, respectively. N, nucleus; C, cytoplasm.

9. K

   Genome‐wide effect of ALYREF overexpression on nuclear RNA levels. According to nuclear rRNA‐depleted RNA‐seq data shown in Fig 1, the mRNAs and lncRNAs that are accumulated more than 1.5‐fold in the nuclei of ALYREF overexpression cells were grouped into three classes.

10. L

    Screenshots of RNA‐seq signals of PPP1R15A and HSP90AA1 mRNAs are shown as examples as exosome targets that are stabilized in the nucleus upon ALYREF overexpression. Replicates of nuclear RNA‐seq signals in control‐ and ALYREF‐overexpressing cells are shown above the gene profile, and those in control, hRRP40, and hMTR4 knockdown cells are shown in below.

Source data are available online for this figure.

Figure EV5. The competition between nuclear degradation and mRNA export

1. A

   Western analysis to examine the overexpression of Flag‐ALYREF.

2. B

   According to the change in nuclear levels upon ALYREF overexpression, the mRNAs and lncRNAs are grouped into three classes. The percentages of RNAs falling in each class in each replicate are shown.

3. C

   The overlap of mRNAs and lncRNAs that are accumulated in the nucleus more than 1.5‐fold upon ALYREF overexpression in two biological replicates.

4. D

   Western analysis to examine the overexpression of Flag‐hMTR4.

5. E

   Western blotting to examine UAP56 and hRRP40 knockdown efficiencies. Tubulin serves as the loading control.

6. F, G

   Blocking mRNA export enhances hMTR4 recruitment to exosome target RNAs. Cntl/hRRP40 or UAP56/URH49/hRRP40 siRNA‐treated HeLa cells were used for IPs using the hMTR4 antibody. The immunoprecipitates were subjected to Western analysis to detect hMTR4 (F) and RT–qPCRs to detect immunoprecipitated RNAs (G). Error bars, standard deviations (n = 3). Statistical analysis was performed using Student's t‐test. *P < 0.05, **P < 0.01.

Source data are available online for this figure.

Figure 7. hMTR4 functions in controlling balanced nuclear RNA pools for degradation and export

1. A, B

   RNA‐IP analysis shows the association of ALYREF with RNAs is weakened upon hMTR4 overexpression. HEK293 cells were infected with lentivirus expressing Flag‐DDX3 (control) or Flag‐hMTR4. IPs were carried out using the ALYREF antibody. The immunoprecipitates were used for RT–qPCRs to detect immunoprecipitated RNAs (A) and Western analysis to detect immunoprecipitated ALYREF (B). Statistical analysis was performed using Student's t‐test. Error bars, standard deviations (n = 3). *P < 0.05, ***P < 0.001.

2. C

   Western blotting to examine ALYREF/THOC2 knockdown efficiency. Tubulin serves as the loading control.

3. D, E

   Co‐knockdown of ALYREF and hTHO enhances hMTR4 recruitment to exosome target RNAs. Control or ALYREF/THOC2 siRNA‐treated HeLa cells were used for IPs with the hMTR4 antibody. The immunoprecipitates were subjected to RT–qPCRs to detect immunoprecipitated RNAs (D) and Western analysis to detect hMTR4 (E). Statistical analysis was performed using Student's t‐test. Error bars, standard deviations (n = 3). *P < 0.05, **P < 0.01.

4. F

   Western blotting to examine the purity of the cytoplasmic fractions. CBP80 and tubulin were used as nuclear and cytoplasmic markers, respectively. C, cytoplasm; W, whole cell.

5. G

   RNAs from cytoplasmic fractions prepared in (F) were used for RT–qPCRs to examine the cytoplasmic levels of indicated exosome target RNAs. The relative levels of indicated RNAs to 18S rRNA are quantified and indicated in the graph. Statistical analysis was performed using Student's t‐test. Error bars, standard deviations (n = 3). *P < 0.05, **P < 0.01.

6. H

   Targeting ALYREF to RNAs prevents exosome activity and/or recruitment. (Top) Illustration of the β‐globin reporter construct that contains MS2‐binding site. (Bottom) RT–qPCRs to examine the levels of β‐globin cDNA‐6MS2 co‐transfected with Flag‐MS2‐MBP or Flag‐MS2‐ALYREF expression constructs in normal HeLa cells (left graph) or in control and hMTR4 knockdown cells (right graph). Error bars, standard deviations (n = 3). Statistical analysis was performed using Student's t‐test. **P < 0.01.

7. I

   Model for the role of hMTR4 in exosome recruitment and in maintaining balance nuclear RNA pools for degradation and export. (Left) In normal cells, hMTR4 competes with ALYREF for associating with the CBC complex bound on RNAs and specifically recruits the exosome to RNAs failing to packed in export‐competent RNPs (see the details in the Discussion section). (Middle) In cells with mRNA export defect, hMTR4 gains access to more RNAs that are subject to degradation in the nucleus. (Right) In hMTR4 downregulated cells, nuclear RNA degradation is blocked and ALYREF, properly together with other mRNA export factors, associates with more RNAs that are subsequently exported to the cytoplasm.

Source data are available online for this figure.