Journal: Genes & Development

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

doi: 10.1101/gad.302604.117

Figure Lengend Snippet: 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.

Article Snippet: Mtr4 (NB100-1574), ZCCHC8 (NB100-94995), ZCCHC7 (NBP1-89175), Dis3 (H00022894-B01P), and Rrp6 (NBP1-32870) antibodies were from Novus Biologicals.

Techniques: Western Blot, Transfection, Control, Knockdown, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: Genes & Development

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

doi: 10.1101/gad.302604.117

Figure Lengend Snippet: 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 .

Article Snippet: Mtr4 (NB100-1574), ZCCHC8 (NB100-94995), ZCCHC7 (NBP1-89175), Dis3 (H00022894-B01P), and Rrp6 (NBP1-32870) antibodies were from Novus Biologicals.

Techniques: Western Blot, Stable Transfection, Expressing, Filtration, Sequencing, RNA Binding Assay

Journal: Genes & Development

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

doi: 10.1101/gad.302604.117

Figure Lengend Snippet: 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.

Article Snippet: Mtr4 (NB100-1574), ZCCHC8 (NB100-94995), ZCCHC7 (NBP1-89175), Dis3 (H00022894-B01P), and Rrp6 (NBP1-32870) antibodies were from Novus Biologicals.

Techniques: Stable Transfection, Expressing, Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay, Knockdown, Quantitative RT-PCR, Transfection

Journal: Genes & Development

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

doi: 10.1101/gad.302604.117

Figure Lengend Snippet: 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.

Article Snippet: Mtr4 (NB100-1574), ZCCHC8 (NB100-94995), ZCCHC7 (NBP1-89175), Dis3 (H00022894-B01P), and Rrp6 (NBP1-32870) antibodies were from Novus Biologicals.

Techniques: Transfection, SDS Page, Software

Journal: Genes & Development

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

doi: 10.1101/gad.302604.117

Figure Lengend Snippet: 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.

Article Snippet: Mtr4 (NB100-1574), ZCCHC8 (NB100-94995), ZCCHC7 (NBP1-89175), Dis3 (H00022894-B01P), and Rrp6 (NBP1-32870) antibodies were from Novus Biologicals.

Techniques: Knockdown, Western Blot, Fractionation, Isolation, Synthesized, Amplification, Random Primed

Journal: Genes & Development

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

doi: 10.1101/gad.302604.117

Figure Lengend Snippet: 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.

Article Snippet: Mtr4 (NB100-1574), ZCCHC8 (NB100-94995), ZCCHC7 (NBP1-89175), Dis3 (H00022894-B01P), and Rrp6 (NBP1-32870) antibodies were from Novus Biologicals.

Techniques: Transfection, Control, cDNA Synthesis, Reverse Transcription Polymerase Chain Reaction

Journal: Genes & Development

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

doi: 10.1101/gad.302604.117

Figure Lengend Snippet: 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.

Article Snippet: Mtr4 (NB100-1574), ZCCHC8 (NB100-94995), ZCCHC7 (NBP1-89175), Dis3 (H00022894-B01P), and Rrp6 (NBP1-32870) antibodies were from Novus Biologicals.

Techniques: Disruption

Journal: bioRxiv

Article Title: KSHV promotes oncogenic FOS to inhibit nuclease AEN and transactivate RGS2 for AKT phosphorylation

doi: 10.1101/2024.01.27.577582

Figure Lengend Snippet: (A) MTR4 knockdown (KD) by MTR4-targeting siRNA (siMTR4) promotes FOS protein expression in HEK293T cells. The effect of MTR4 KD on FOS expression in HEK293T cells was examined by Western blotting at 48 h after transfection. A non-targeting siRNA (siNT) served as a siRNA control and GAPDH as a sample loading control. (B) KSHV lytic infection and ORF57 expression do not affect MTR4 expression. MTR4 RNA expression was quantified by RT-qPCR in BCBL-1 with latent and lytic infection and in HEK293T cells transfected with an ORF57-expressing (ORF57) or an empty vector control (Ctrl) vector. Data from all three separate experiments, each with three replicates, were averaged with mean ± SD. NS – no significance, two-tailed Student t -test. (C) No protein-protein interaction between ORF57 and MTR4. Co-immunoprecipitation of ORF57 and MTR4 proteins from total cell extracts from HEK293T cells transfected with an ORF57-expressing vector was performed by using rabbit anti-ORF57 or anti-MTR4 antibodies, with corresponding species-specific IgG isotype serving as an antibody control. ORF57 and MTR4 proteins were immunoblotted with the corresponding antibodies as indicated.

Article Snippet: Mouse polyclonal anti-AEN was from Millipore Sigma, rabbit polyclonal anti-RGS2 from Abcam, rabbit polyclonal anti-ZFC3H1(MTR4) antibody from Novus biologicals, and rabbit IgG isotype, mouse IgG isotype were from Thermo Fisher Scientific.

Techniques: Expressing, Western Blot, Transfection, Infection, RNA Expression, Quantitative RT-PCR, Plasmid Preparation, Two Tailed Test, Immunoprecipitation

Journal: Cell Reports

Article Title: Dual agonistic and antagonistic roles of ZC3H18 provide for co-activation of distinct nuclear RNA decay pathways

doi: 10.1016/j.celrep.2023.113325

Figure Lengend Snippet: The ZFC3H1 N terminus harbors important information for its function in RNA decay (A) Left: schematic representation of the PAXT connection, highlighting its ZFC3H1-MTR4 core. See text for more detail. Right: experimental design of the ZFC3H1 mutational analysis where the endogenous protein was replaced by one of the MYC-tagged variants from (B). (B) Schematic map of full-length ZFC3H1 (Z1 FL ), depicting its predicted domains: SER, serine-rich; CC, coiled coil; PRO, proline-rich; ZnF, zinc finger; TPR, tetratricopeptide repeat and the regions of ZFC3H1 covered by the generated Z1 variants. (C) qRT-PCR analysis of selected PAXT substrates (proASH1L, proSAMD4B, FOXD2-AS1) and a negative control NEXT substrate (proSTAT3) from total RNA isolated from HeLa cells treated with control (siLUC) or ZFC3H1-targeting siRNA, while stably expressing one of the siRNA-immune ZFC3H1 variants from (B). The RT step was performed using a mixture of random and oligo d(T) 20 VN primers. qPCR amplicons were positioned in TU 5′-end regions. Results were normalized to 18S rRNA levels and plotted as fold change relative to siLUC control samples. Columns represent the mean values of three technical replicates, which are depicted as individual data points, with error bars showing the standard deviation (SD). (D) Top: schematic representation of the mouse Z1 1-1363 variant and its N-terminal truncations used for further characterization of ZFC3H1. Bottom: western blotting (WB) analysis of expression levels of mouse ZFC3H1 variants in mZfc3h1 −/− mESCs, using antibodies against ZFC3H1 and vinculin (VCL) as a loading control. (E) qRT-PCR analysis of selected mouse PAXT substrates and OCT4 mRNA (negative control) from total RNA isolated from cells used in (D). Results were normalized to GAPDH mRNA levels and plotted as fold changes relative to WT control samples. Data representation as in (C).

Article Snippet: Rabbit polyclonal anti-MTR4 (SKIV2L2) , Abcam , Cat# ab70551; RRID: AB_1270701.

Techniques: Generated, Quantitative RT-PCR, Negative Control, Isolation, Stable Transfection, Expressing, Standard Deviation, Variant Assay, Western Blot

Journal: Cell Reports

Article Title: Dual agonistic and antagonistic roles of ZC3H18 provide for co-activation of distinct nuclear RNA decay pathways

doi: 10.1016/j.celrep.2023.113325

Figure Lengend Snippet: ZFC3H1 and ZC3H18 compete for ARS2 binding (A) WB analysis of MYC IPs from lysates of HeLa cells stably expressing MYC-ARS2 and treated with either control siRNA (siLUC) or siZC3H18. Input and IP samples were probed with antibodies against MYC, ZC3H18, ZCCHC8, and ZFC3H1 as indicated. (B) WB analysis of MYC IPs from lysates of HeLa cells expressing MYC-tagged ZC3H18 WT , MYC-tagged ZC3H18 MUT , and untagged control cells. Input and IP samples were probed with antibodies against MYC, ARS2, NCBP2, ZCCHC8, MTR4, and tubulin alpha (TUBA) as a control. The membrane probed for ZCCHC8 was incompletely stripped and subsequently probed for MTR4 resulting in detection of residual ZCCHC8 signal as indicated. (C) Schematic representation of the suggested inhibition of a common ZFC3H1/ZC3H18 binding site on ARS2 by overexpression of Z1 1-209(WT) . The zoom-ins depict the conserved ARS2-binding SLiM in the indicated proteins and the corresponding binding site on ARS2. (D) qRT-PCR analysis as in Figure 4 E but of total RNA isolated from WT HeLa control cells or from cells following 2 days of overexpression of stably integrated Z1 1-209(WT) or Z1 1-209(MUT) . (E) Violin boxplots depicting log 2 fold change distribution of PAXT- (left) and NEXT-sensitive (right) RNAs in a total RNA-seq dataset generated from cells used in (D). Both RNA groups were further categorized into ARS2 dependent and independent as in Figure 3 A. p values, calculated as in Figure 3 A, indicate the difference between ARS2-dependent and -independent groups. (F) Heatmaps displaying log 2 fold changes of 3′ end 4-thiouridine RNA-seq data from siZFC3H1 (left) and siZCCHC8 (right) samples relative to their control. Data were plotted in 2-kb regions centered around annotated TESs of the top 50% of ARS2-dependent PAXT- and NEXT-sensitive TUs. The average signals from three replicates are shown. RNA samples were in vitro polyadenylated to capture both pA + and pA − transcripts. (G) Genome browser views of representative TUs from (F). Tracks contain the average signal of three biological replicate samples showing the relevant strand and genomic coordinates. The in-house annotation from Figure 3 D is displayed for the relevant strand below the tracks.

Article Snippet: Rabbit polyclonal anti-MTR4 (SKIV2L2) , Abcam , Cat# ab70551; RRID: AB_1270701.

Techniques: Binding Assay, Stable Transfection, Expressing, Membrane, Inhibition, Over Expression, Quantitative RT-PCR, Isolation, RNA Sequencing Assay, Generated, In Vitro

Journal: Cell Reports

Article Title: Dual agonistic and antagonistic roles of ZC3H18 provide for co-activation of distinct nuclear RNA decay pathways

doi: 10.1016/j.celrep.2023.113325

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-MTR4 (SKIV2L2) , Abcam , Cat# ab70551; RRID: AB_1270701.

Techniques: Virus, Recombinant, Transfection, SYBR Green Assay, Protease Inhibitor, Clone Assay, Purification, Magnetic Beads, Hydrophilic Interaction Liquid Chromatography, Software

Journal: Molecular Cell

Article Title: Mammalian RNA Decay Pathways Are Highly Specialized and Widely Linked to Translation

doi: 10.1016/j.molcel.2020.01.007

Figure Lengend Snippet: Mammalian mRNA Decay Pathways Target Distinct Transcripts (A) RNA decay pathways. (B) CRAC outline. (C and D) PCA (C) and correlation matrix (D) based on decay factor binding (CRAC counts) to mRNAs. Replicates correspond to separate experiments for the same cell line. (E) t-SNE representation of mRNAs based on relative binding to MTR4, SKIV2L, and XRN1. (F and G) CRAC coverage across individual mRNAs. Transcripts in (F) illustrate different XRN1:SKIV2L ratios, whereas (G) depicts transcripts highlighed in panel (I). (H) Conditional knockout strategy for Skiv2l . (I) Differential expression analysis for Skiv2l knockout for the mRNAs in (E), with significantly changing transcripts (DESeq2 padj < 0.05) colored by SKIV2L binding (as in E). See also Figure S1 and , , and .

Article Snippet: The following primary antibodies were used for western blotting: rat anti-HA (1:1,000, Roche 11867423001), mouse anti-Flag (1:1,000, Sigma clone M2), rabbit anti-Mtr4 (1:1,000, Thermo Fisher PA5-57927).

Techniques: Binding Assay, Knock-Out, Expressing

Journal: Molecular Cell

Article Title: Mammalian RNA Decay Pathways Are Highly Specialized and Widely Linked to Translation

doi: 10.1016/j.molcel.2020.01.007

Figure Lengend Snippet: SKIV2L Binding and 3′–5′ Decay Increase upon Aven Knockout (A and B) CRAC signal for AVEN around start and stop codons (A) and on the ribosomal 40S subunit (B). (C) PCA based on mRNA counts. Shapes indicate different clones. (D) CRAC coverage for individual mRNAs. (E) SKIV2L CRAC around start and stop codons in WT and Aven −/− cells. (F) Changes in SKIV2L CRAC binding (left) and RNA-seq counts (right) for Aven −/− versus WT cells. Significantly up/downregulated transcripts (padj < 0.05) are colored by AVEN CRAC counts in WT cells, relative to SKIV2L+XRN1+MTR4 counts, and replication-dependent histone mRNAs are circled. (G) Proportion of AVEN or SKIV2L CRAC reads in mRNAs with 3′ U-tails. See also Figure S3 and .

Article Snippet: The following primary antibodies were used for western blotting: rat anti-HA (1:1,000, Roche 11867423001), mouse anti-Flag (1:1,000, Sigma clone M2), rabbit anti-Mtr4 (1:1,000, Thermo Fisher PA5-57927).

Techniques: Binding Assay, Knock-Out, Clone Assay, RNA Sequencing Assay

Journal: Molecular Cell

Article Title: Mammalian RNA Decay Pathways Are Highly Specialized and Widely Linked to Translation

doi: 10.1016/j.molcel.2020.01.007

Figure Lengend Snippet:

Article Snippet: The following primary antibodies were used for western blotting: rat anti-HA (1:1,000, Roche 11867423001), mouse anti-Flag (1:1,000, Sigma clone M2), rabbit anti-Mtr4 (1:1,000, Thermo Fisher PA5-57927).

Techniques: Recombinant, Transfection, Protease Inhibitor, Autoradiography, Gel Extraction, Western Blot, RNA Extraction, SYBR Green Assay, Sequencing, Software