Journal: The Journal of Biological Chemistry

Article Title: The putative RNA helicase DDX1 associates with the nuclear RNA exosome and modulates RNA/DNA hybrids (R-loops)

doi: 10.1016/j.jbc.2024.105646

Figure Lengend Snippet: RNA exosome subunits coimmunoprecipitate with tagged EXOSC3. A , the RNA exosome is a conserved exo/endoribonuclease complex that comprises ten subunits. Nine of the ten subunits are structural and termed exosome components. EXOSC1, EXOSC2, and EXOSC3 make up the cap and EXOSC4, EXOSC5, EXOSC6, EXOSC7, EXOSC8, and EXOSC9 make up a barrel-shaped core . In this graphic, EXOSC3 ( navy ) and EXOSC9 ( green ) are highlighted . EXOSC6, EXOSC7, and EXOSC8 are positioned behind subunits EXOSC4, EXOSC5, and EXOSC9, and consequently are not visible. The catalytic subunit, DIS3 or DIS3L, sits at the base of the complex [PDB 6H25 ]. B , EXOSC9 core subunit coprecipitates with myc-EXOSC3 from murine neuronal N2A cell line. Cells were transfected with a plasmid encoding Vector control or myc-EXOSC3, followed by immunoprecipitation using anti-myc magnetic beads. Input for Vector control and myc-EXOSC3 was probed by an anti-myc antibody and a band corresponding to the molecular weight is detected in the input but not Vector control for myc-EXOSC3. Input for Vector control and myc-EXOSC3 is probed by an anti-EXOSC9 antibody and a band at the corresponding molecular weight is present in both lanes. Bound for Vector control and myc-EXOSC3 is probed by an anti-myc and an anti-EXOSC9 antibody, and a band corresponding to the molecular weight is detected in the bound fraction for myc-EXOSC3 but not for Vector control. Stain-free blot indicates the loading of total protein in the input. Immunoprecipitation of the myc-tagged EXOSC3 copurifies with the endogenous EXOSC9 subunit. C , eluates of the bound myc-EXOSC3 immunoprecipitation were analyzed by LC-tandem mass spectrometry. A table shows all RNA exosome subunits detected, listing the peptide-spectrum matches (PSM) and the peptide numbers for each subunit. Vector IP serves as a control. IP, immunoprecipitation.

Article Snippet: Studies in Schizosaccharomyces pombe have revealed PTMs in Dis3, Mtr4, Rrp40 (EXOSC3), Rrp43 (EXOSC8), and Rrp46 (EXOSC5), though only PTM mimetics in Dis3 and Mtr4 impacted RNA processing ( ).

Techniques: Transfection, Plasmid Preparation, Control, Immunoprecipitation, Magnetic Beads, Molecular Weight, Staining, Mass Spectrometry

Journal: Nucleic Acids Research

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

doi: 10.1093/nar/gky650

Figure Lengend Snippet: 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.

Article Snippet: The rabbit polyclonal antibodies against MTR4 and MTR3 were purchased from ABclonal Technology.

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Knockdown, Control, Staining, Marker, Mutagenesis, Functional Assay, Transfection, Expressing, Plasmid Preparation, Confocal Microscopy

Journal: Nucleic Acids Research

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

doi: 10.1093/nar/gky650

Figure Lengend Snippet: 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.

Article Snippet: The rabbit polyclonal antibodies against MTR4 and MTR3 were purchased from ABclonal Technology.

Techniques: Western Blot, Knockdown, Control, Construct, Injection, Marker, Transfection, Staining

Journal: Nucleic Acids Research

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

doi: 10.1093/nar/gky650

Figure Lengend Snippet: 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.

Article Snippet: The rabbit polyclonal antibodies against MTR4 and MTR3 were purchased from ABclonal Technology.

Techniques: Western Blot, Knockdown, Transfection, Control, Marker, Staining, RNA Sequencing

Journal: Nucleic Acids Research

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

doi: 10.1093/nar/gky650

Figure Lengend Snippet: 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.

Article Snippet: The rabbit polyclonal antibodies against MTR4 and MTR3 were purchased from ABclonal Technology.

Techniques: Knockdown, Control, Genome Wide, Staining, Marker, Confocal Microscopy

Journal: Nucleic Acids Research

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

doi: 10.1093/nar/gky650

Figure Lengend Snippet: 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.

Article Snippet: The rabbit polyclonal antibodies against MTR4 and MTR3 were purchased from ABclonal Technology.

Techniques: Western Blot, Control, Knockdown, Transfection, Staining, Marker, Quantitative RT-PCR, Confocal Microscopy

Journal: Nucleic Acids Research

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

doi: 10.1093/nar/gky650

Figure Lengend Snippet: 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.

Article Snippet: The rabbit polyclonal antibodies against MTR4 and MTR3 were purchased from ABclonal Technology.

Techniques: Control, Knockdown, Confocal Microscopy, Construct, Injection, Plasmid Preparation, Marker, Staining

Journal: Nucleic Acids Research

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

doi: 10.1093/nar/gky650

Figure Lengend Snippet: 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.

Article Snippet: The rabbit polyclonal antibodies against MTR4 and MTR3 were purchased from ABclonal Technology.

Techniques: Binding Assay

Journal: Parasitology

Article Title: Nuclear mRNA maturation and mRNA export control: from trypanosomes to opisthokonts

doi: 10.1017/S0031182021000068

Figure Lengend Snippet: Overview of nuclear mRNA metabolism. This diagram summarizes the basic steps of nuclear mRNA processing in a simplified version; all steps are discussed in the review in greater detail. Note that not all components are present in all organisms. For clarity, I have sorted processing steps in a pathway, but the order of events is not fully understood and processing steps can also occur in different orders or simultaneously. Abbreviations: CTD: C-terminal domain; CBC: cap binding complex; SR: serine−arginine rich; RNA PolII: RNA polymerase II; CFP: cleavage and polyadenylation factor; TREX: couples tr anscription and ex port; TRAMP: Trf4-Air2-Mtr4 polyadenylation.

Article Snippet: One is the TRAMP complex (Trf4-Air2-Mtr4 polyadenylation), that consists of the RNA helicase Mtr4 (MTR4 (SKIV2L2) in human), the poly(A) polymerase Trf4 (PAPD5 (TRF4-2) in human) and the RNA binding protein Air1 (ZCCHC7 (AIR1) in human) (Schmid and Jensen, ) ( ).

Techniques: Binding Assay

Journal: Parasitology

Article Title: Nuclear mRNA maturation and mRNA export control: from trypanosomes to opisthokonts

doi: 10.1017/S0031182021000068

Figure Lengend Snippet: Exosome and TRAMP complex subunits in trypanosomes and yeast

Article Snippet: One is the TRAMP complex (Trf4-Air2-Mtr4 polyadenylation), that consists of the RNA helicase Mtr4 (MTR4 (SKIV2L2) in human), the poly(A) polymerase Trf4 (PAPD5 (TRF4-2) in human) and the RNA binding protein Air1 (ZCCHC7 (AIR1) in human) (Schmid and Jensen, ) ( ).

Techniques:

Journal: Nature Communications

Article Title: The MTR4/hnRNPK complex surveils aberrant polyadenylated RNAs with multiple exons

doi: 10.1038/s41467-024-51981-8

Figure Lengend Snippet: a Schematic illustration of the identification of RNAs destabilized by MTR4. (left) To identify transcript variant repertoire upregulated upon MTR4 depletion, HeLa cells transfected with siCont or siMTR4 were subjected to short- and long-read sequencing, as presented in Supplementary Fig. . Pale-colored boxes are transcripts identified in this study. (right) To identify the transcript variant stabilized by MTR4 depletion, HeLa cells transfected with siCont or siMTR4 were treated with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), a transcription inhibitor, for the indicated times and then subjected to 3′-sequencing to estimate stability. By combining these data, MTR4-target transcripts were determined. b Schematic representation of two types of 3′ e X tended T ranscript (3XT) and 3′ e X tended R egions (3XRs). The middle exon is the exon other than the first or last exon in annotated transcripts. 3XTs are transcripts that have an extended last exon with (multi-exon 3XT) or without (mono-exon 3XT) splicing events. c A violin plot from NanoBlot results displaying the distribution of sequenced read lengths of a representative 3XT. ATP23 (left, blue) and HECTD2 (right, red) are examples of genes with mono- and multi- exon 3XTs, respectively. Arrowheads indicate the lengths of ATP23 (blue) and HECTD2 (red) 3XT. d qRT-PCR analysis of mono- (blue) and multi-exon (red) 3XT expression in HeLa cells transfected with siRNA targeting MTR4 . Results are expressed as the mean ± s.d. (n = 3 biologically independent replicates). Paired two-sided Student’s t-test; * p < 0.05. The exact p -values are ATP23 3XT: p = 0.00916 (siCont vs siMTR4#1), p = 0.00074 (siCont vs siMTR4#2), TP53TG1 3XT: p = 0.00077 (siCont vs siMTR4#1), p = 0.00227 (siCont vs siMTR4#2), USP45 3XT: p = 0.00792 (siCont vs siMTR4#1), p = 0.01991 (siCont vs siMTR4#2), HECTD2 3XT: p = 0.00441 (siCont vs siMTR4#1), p = 0.00667 (siCont vs siMTR4#2), SPRED2 3XT: p = 0.00794 (siCont vs siMTR4#1), p = 0.00122 (siCont vs siMTR4#2), KCTD13 3XT: p = 0.02597 (siCont vs siMTR4#1), p = 0.02677 (siCont vs siMTR4#2). Source data are provided as a Source Data file.

Article Snippet: siRNA duplexes targeting MTR4 were purchased from Hokkaido System Science (HSS).

Techniques: Variant Assay, Transfection, Sequencing, Quantitative RT-PCR, Expressing

Journal: Nature Communications

Article Title: The MTR4/hnRNPK complex surveils aberrant polyadenylated RNAs with multiple exons

doi: 10.1038/s41467-024-51981-8

Figure Lengend Snippet: a – d (left) qRT-PCR analysis of mono- (blue) and multi-exon (red) 3XT expression in HeLa cells transfected with siRNA targeting EXOSC5 ( a ), PABPN1 ( b ), RBM7 ( c ) or ZCCHC7 ( d ). qRT-PCR results are expressed as the mean ± s.d. ( n = 3 biologically independent replicates). Paired two-sided Student’s t-test; * p < 0.05. (middle) qRT-PCR analysis of EXOSC5 , PABPN1 or RBM7 expression in HeLa cells transfected with siRNA targeting EXOSC5 ( a ), PABPN1 ( b ), RBM7 ( c ), or ZCCHC7 ( d ). qRT-PCR results are expressed as the mean ± s.d. (n = 3 biologically independent replicates). Paired two-sided Student’s t-test; * p < 0.05. (right) Cell lysates from HeLa cells transfected with siRNA targeting the indicated genes were subjected to immunoblotting analysis with anti-EXOSC5 ( a ), anti-PABPN1 ( b ), a nti-RBM7 ( c ), anti-ZCCHC7 ( d ), or anti-GAPDH antibodies. GAPDH was used as a loading control. The exact p -values are a ATP23 3XT: p = 0.00833 (siCont vs siEXOSC5#1), p = 0.00927 (siCont vs siEXOSC5#2), TP53TG1 3XT: p = 0.00048 (siCont vs siEXOSC5#1), p = 0.00067 (siCont vs siEXOSC5#2), USP45 3XT: p = 0.00187 (siCont vs siEXOSC5#1), p = 0.00312 (siCont vs siEXOSC5#2), HECTD2 3XT: p = 0.00187 (siCont vs siEXOSC5#1), p = 0.00312 (siCont vs siEXOSC5#2), SPRED2 3XT: p = 0.00977 (siCont vs siEXOSC5#1), p = 0.02162 (siCont vs siEXOSC5#2), KCTD13 3XT: p = 0.00443 (siCont vs siEXOSC5#1), p = 0.00792 (siCont vs siEXOSC5#2), EXOSC5 : p = 0.00010 (siCont vs siEXOSC5#1), p = 0.00007 (siCont vs siEXOSC5#2); b ATP23 3XT: p = 0.00054 (siCont vs siPABPN1#1), p = 0.01888 (siCont vs siPABPN1#2), TP53TG1 3XT: p = 0.02140 (siCont vs siPABPN1#1), p = 0.04520 (siCont vs siPABPN1#2), USP45 3XT: p = 0.00101 (siCont vs siPABPN1#1), p = 0.00695 (siCont vs siPABPN1#2), HECTD2 3XT: p = 0.00956 (siCont vs siPABPN1#1), p = 0.03642 (siCont vs siPABPN1#2), SPRED2 3XT: p = 0.01983 (siCont vs siPABPN1#1), p = 0.01966 (siCont vs siPABPN1#2), KCTD13 3XT: p = 0.01838 (siCont vs siPABPN1#1), p = 0.03199 (siCont vs siPABPN1#2), PABPN1 : p = 0.00088 (siCont vs siPABPN1#1), p = 0.00021 (siCont vs siPABPN1#2); c RBM7 : p = 0.00033 (siCont vs siRBM7#1), p = 0.00006 (siCont vs siRBM7#2); d ZCCHC7 : p = 7.8721E-07 (siCont vs si ZCCHC7 #1), p = 0.00009 (siCont vs si ZCCHC7 #2). Source data are provided as a Source Data file.

Article Snippet: siRNA duplexes targeting MTR4 were purchased from Hokkaido System Science (HSS).

Techniques: Quantitative RT-PCR, Expressing, Transfection, Western Blot, Control

Journal: Nature Communications

Article Title: The MTR4/hnRNPK complex surveils aberrant polyadenylated RNAs with multiple exons

doi: 10.1038/s41467-024-51981-8

Figure Lengend Snippet: a The C-rich motif is enriched in MTR4-target multi-exon 3XRs. The table shows the results of MEME motif enrichment analysis. E-value; the expected number of motifs with the given log likelihood ratio (or higher), and with the same width and site count that one would find in a similarly sized set of random sequences. Sites; the number of sites contributing to the construction of the motif. b Venn diagram showing the CCWSCC-matched RNA-binding proteins speculated by Tomtom using different distance metrics. Pearson; Pearson correlation coefficient. Euclidean; Euclidean distance. Sandelin; Sandelin-Wasserman similarity. W; A or T. S; G or C. c qRT-PCR analysis of mono- (blue) and multi-exon (red) 3XT expression in HeLa cells transfected with si-hnRNPK. Results are expressed as the mean ± s.d. ( n = 3 biologically independent replicates). Paired two-sided Student’s t-test; * p < 0.05. d Lysates from HeLa cells were subjected to immunoprecipitation with an anti-hnRNPK antibody or rabbit IgG, followed by qRT-PCR analysis to detect the indicated RNAs. SNHG9 and SNHG19 RNA were used as negative controls. Results are expressed as the mean ± s.d. (n = 3 biologically independent replicates). Paired two-sided Student’s t-test; * p < 0.05. e hnRNPK associates with MTR4 in vivo. Lysates from HeLa cells were subjected to immunoprecipitation with an anti-MTR4 antibody, an anti-hnRNPK antibody or rabbit IgG followed by immunoblotting analysis with anti-MTR4, anti-hnRNPK or anti-GAPDH antibodies. GAPDH was used as a negative control. This experiment was repeated independently two times with similar results. The exact p -values are c HECTD2 3XT: p = 0.03689 (siCont vs si-hnRNPK#1), p = 0.04965 (siCont vs si-hnRNPK2), SPRED2 3XT: p = 0.01871 (siCont vs si-hnRNPK#1), p = 0.02218 (siCont vs si-hnRNPK#2), KCTD13 3XT: p = 0.01953 (siCont vs si-hnRNPK#1), p = 0.04751 (siCont vs si-hnRNPK#2); d HECTD2 3XT: p = 0.01229 (rabbit IgG vs hnRNPK IP), SPRED2 3XT: p = 0.00157 (rabbit IgG vs hnRNPK IP), KCTD13 3XT: p = 0.01079 (rabbit IgG vs hnRNPK IP). Source data are provided as a Source Data file.

Article Snippet: siRNA duplexes targeting MTR4 were purchased from Hokkaido System Science (HSS).

Techniques: RNA Binding Assay, Quantitative RT-PCR, Expressing, Transfection, Immunoprecipitation, In Vivo, Western Blot, Negative Control

Journal: Nature Communications

Article Title: The MTR4/hnRNPK complex surveils aberrant polyadenylated RNAs with multiple exons

doi: 10.1038/s41467-024-51981-8

Figure Lengend Snippet: A schematic model showing the mechanism of multi-exon 3XT degradation by the hnRNPK-MTR4-RNA exosome complex. MTR4 interacts with hnRNPK, which recognizes 3XRs of multi-exon 3XTs, and degrades them via the RNA exosome complex in the nucleus of cells under normal conditions. RNA surveillance dysregulation, in which MTR4/hnRNPK is depleted, leads to the production of multi-exon 3XT-derived proteins, resulting in the formation of aberrant biomolecular condensates.

Article Snippet: siRNA duplexes targeting MTR4 were purchased from Hokkaido System Science (HSS).

Techniques: Derivative Assay