An Mtr4/ZFC3H1 complex facilitates turnover of unstable nuclear RNAs to prevent their cytoplasmic transport and global translational repression

Abstract

Many long noncoding RNAs (lncRNAs) are unstable and rapidly degraded in the nucleus by the nuclear exosome. An exosome adaptor complex called NEXT (nuclear exosome targeting) functions to facilitate turnover of some of these lncRNAs. Here we show that knockdown of one NEXT subunit, Mtr4, but neither of the other two subunits, resulted in accumulation of two types of lncRNAs: prematurely terminated RNAs (ptRNAs) and upstream antisense RNAs (uaRNAs). This suggested a NEXT-independent Mtr4 function, and, consistent with this, we isolated a distinct complex containing Mtr4 and the zinc finger protein ZFC3H1. Strikingly, knockdown of either protein not only increased pt/uaRNA levels but also led to their accumulation in the cytoplasm. Furthermore, all pt/uaRNAs examined associated with active ribosomes, but, paradoxically, this correlated with a global reduction in heavy polysomes and overall repression of translation. Our findings highlight a critical role for Mtr4/ZFC3H1 in nuclear surveillance of naturally unstable lncRNAs to prevent their accumulation, transport to the cytoplasm, and resultant disruption of protein synthesis.

Keywords: Mtr4; ZFC3H1; exosome; lncRNA; polyadenylation.

Figure 1.

Global analysis of poly(A)⁺ transcript levels following depletion of individual NEXT subunits. (A) Western blot analysis of HeLa cell extracts after 48 h of transfection of control (Ctrl), Mtr4, RBM7, or ZCCHC8 siRNA. (B) RBM7 mRNA level after 48 h of siRBM7 treatment. RBM7 mRNA was normalized to GAPDH mRNA, and the normalized levels in siCtrl-treated cells were set to 1. Bars represent mean ± SD. n = 3. An asterisk denotes significant difference from siCtrl (P < 0.05) using an unpaired Student's t-test. (C) Schematic of different transcript types analyzed: transcripts using the first (F), middle (M), or last (L) potential PAS in the 3′ untranslated region (UTR); the single (S; no 3′ UTR APA) PAS in the 3′ UTR; the intronic PAS in the composite terminal exon (Ic); the intronic PAS in the skipped terminal exon (Is); the upstream (not 3′-most) exonic PAS (E); and the upstream antisense transcripts (UA). (D) Changes in relative abundance of the indicated transcript types following knockdown of indicated NEXT subunits. The percentage of genes showing increases (UP) or decreases (DWN) of each type of transcript are indicated. False discovery rate < 0.05. (E) Metagene plots of ptRNAs and uaRNAs. Data are presented as strand-specific reads per million (RPM) at PAS positions within 4 kb upstream of or downstream from the transcription start site. (F,H) RT-qPCR [oligo(dT)-primed RT and quantitative PCR (qPCR)] analysis of select ptRNAs and corresponding full-length (FL) mRNAs (F) and uaRNAs (H) after knockdown of the individual NEXT subunits. Analysis of two representative PROMPTs—proRBM39 and proFBX07—is also shown in H. Values were normalized to GAPDH mRNA, and the normalized levels in siCtrl-treated cells were set to 1. Bars represent mean ± SD. n = 3. Asterisks denote significant difference from siCtrl (P < 0.05) using an unpaired Student's t-test. (G) Diagram of a ptRNA-producing gene and primers used for RT-qPCR. Arrows indicate the positions of primer targeting sites to analyze ptRNA and full-length mRNA.

Figure 2.

Identification of Mtr4-interacting proteins by cofractionation and MS. (A) Western blotting analysis of HEK293 cells and HEK293 cells stably expressing 3Flag-Mtr4. (Top panel) Blotted with anti-Mtr4 antibodies. (Bottom panel) Blotted with anti-Flag antibodies. (B) Fractions from Superose 6 gel filtration 3Flag-Mtr4-expressing HEK293 cells were analyzed by Western blotting using antibodies against proteins shown at the right. Approximate molecular sizes are indicated at the top, and fractions pooled are indicated at the bottom. (C) Selected proteins copurified with 3Flag-Mtr4 in the indicated pools. Spectral counts and sequence coverage of known Mtr4-interacting partners (NEXT, exosome, and NRDE2), proteins detected as complexes (e.g., NuRD and spliceosome), and RNA processing or RNA-binding proteins are shown. A full protein list is in Supplemental Table S1.

Figure 3.

Mtr4-associated ZFC3H1 is required for down-regulation of ptRNAs and uaRNAs but not NEXT substrates. (A) Cell extracts prepared from HEK293 cells and HEK293 cells stably expressing 3Flag-Mtr4 were used for immunoprecipitation with anti-Flag antibodies in the presence of benzonase and RNase A followed by Western blotting with the indicated antibodies. (B) Cell extracts prepared from HEK293 cells were used for co-IP experiments with anti-ZFC3H1 in the presence of benzonase and RNase A followed by Western blotting with antibodies against the proteins indicated at the right. (C) Western blot analysis of HeLa cell extracts after 72 h of knockdown treatment with the siRNAs indicated at the top; antibodies against the proteins are indicated at the right. (D,E) RT-qPCR analysis of the indicated ptRNAs (D) and the indicated uaRNAs and NEXT substrates proRBM39 and proBIRC4 (E) after the indicated siRNA transfections. Transcript levels were normalized to GAPDH mRNA, and the normalized levels in siCtrl-treated cells were set to 1. Bars represent mean ± SD. n = 3. Asterisks denote significant difference from siCtrl (P < 0.05) using an unpaired Student's t-test.

Figure 4.

Mtr4 knockdown causes cytoplasmic accumulation of stabilized ptRNAs and uaRNAs. (A) Western blotting of subcellular fractions prepared from HeLa cells. Proteins from whole-cell extract (WCE), cytoplasm (Cyt), nuclear-soluble (Nuc), and nuclear-insoluble chromatin (Chr) fractions were analyzed using antibodies directed against the proteins listed on the right. (B,C) Subcellular fractionation was performed after 72 h of siMtr4 (B) or siZFC3H1 (C) treatment, and total RNAs were isolated from each fraction, as indicated at the top. cDNA was synthesized using random or oligo(dT) primer, and the indicated transcripts (shown at the left) were analyzed by PCR. Gels were prestained with ethidium bromide (EtBr). RPPH1 and NEAT1 RNAs were amplified using random-primed RT products and served as cytoplasmic and nuclear-insoluble markers. Other RNAs were amplified using oligo(dT)-primed RT products.

Figure 5.

Cytoplasmic ptRNAs and uaRNAs associate with active ribosomes but lead to reduced global translation. (A) UV absorption profiles at 254 nm of 15%–45% sucrose gradients. HeLa cells were transfected with either control siRNA (siCtrl) or Mtr4 siRNA (siMtr4) for 48 h, and cytoplasmic extracts were prepared from cells with or without 50 µM/mL BTdCPU for 3 h. (B–D) RNAs extracted from each fraction as in A were used for oligo(dT)-primed cDNA synthesis, and the indicated transcripts were analyzed by RT–PCR. Gels were prestained with EtBr. Ribosome/polysome-associated fractions are highlighted with a gray box. An asterisk marks primer dimers.

Figure 6.

Mtr4/ZFC3H1 depletion causes global reduction of translation. (A) Puromycin incorporation assay. HeLa cells transfected with the indicated siRNAs for 48 h were treated with 1 µg/mL puromycin for 30 min. (Lane 3) CHX treatment was performed 10 min prior to puromycin addition. Cell lysates were resolved by SDS-PAGE, and puromycilated proteins were detected using an anti-puromycin antibody. (B) Puromycin-incorporated protein levels as in A were quantitated using LI-COR Image Studio software and normalized by GAPDH levels. The normalized levels in lane 2 were set to 1. Bars represent mean ± SD. n = 3. Asterisks denote significant difference from lane 2 (P < 0.05) using an unpaired Student's t-test.

Figure 7.

Model for the role of the Mtr4/ZFC3H1 complex in the turnover of nuclear polyadenylated transcripts and how its loss affects translation. Model depicting the impact of PPC deficiency on polyadenylated transcriptomes and global translation. Loss of the PPC results in stabilization of ptRNAs and uaRNAs, which are normally rapidly degraded in the nucleus, and these RNAs are then transported to the cytoplasm. The exported RNAs become ribosome-associated and overwhelm the translational machinery, which leads to disruption of the quantitative balance between available ribosomes and translatable RNAs. See the text for details.