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AndersenMass SpectrometryAffinity proteomicsNuclear rna decayRna structureZcchc8-dgcr8 interactionMicroprocessorThe next complexhttps://www.ebi.ac.uk/pride/archive/projects/PXD053925Embryonic Stem CellAffinity enrichment All IPMSs experiments were performed label-free and in triplicates. For whole cell extract IP/MSs, 2x107 cells/IP were standardly extracted in TN extraction buffer (50 mM HEPES pH 7.4, 100 mM NaCl, 0.5% v/v Triton X-100, 0.5% NP-40, 5mM MgCl2, 10% Glycerol) freshly supplemented with 1x protease inhibitors (Roche). Lysates were sheared by sonication (3 x 5 s, amplitude 1, Branson sonifier 250) and cleared by centrifugation at 18,000 rcf for 20 min at 4°C. Clarified lysates were incubated 2 hr at 4ºC with Anti-FLAG magnetic beads prepared with anti-FLAG M2 antibodies (Sigma F3165) conjugated to Dynabeads M-270 Epoxy (Invitrogen). Clarified lysates were incubated 2 hr at 4ºC with anti-FLAG M2 (Sigma, F3165) or Anti-Myc (Abcam, ab32) antibodies and Dynabeads Protein G for Immunoprecipitation (Invitrogen) for IPWB analyses. Beads were washed 3 times with TN extraction buffer, transferring beads to a fresh tube with the final wash. Next, beads were resuspended in TN buffer and either mock-treated or treated with ~ 250 units of Benzonase (Sigma) at 25°C for 20 min in a thermomixer at 1200 rpm. The beads were then washed twice with wash buffer (20 mM Tris/HCl pH8 and 2 mM CaCl2) and proteins were eluted in SDS buffer (2 % SDS, 100 mM Tris pH 6.8, 10 % glycerol) or low pH solution (0.1 M Glycine/HCl pH2.5) at 25°C, shaking for 5 min and adjusted to pH 7-8 by adding 1M Tris/HCl pH8. MS sample preparations were performed with the protein aggregation capture (PAC) procedure employing proteolytic digestion on MagResyn HILIC beads. Proximity labeling with mini-Turbo HA-mTurbo-ZCCHC8 cells were generated by CRISPR/Cas9 Knock-in. In vivo proximity labelling was stimulated by supplementing the media with 50 µM biotin for 1 hour. WT untagged cells were used in parallel as negative controls. Cells were lysed in RIPA-buffer (10 mM Tris pH 7.5, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% v/v Triton X-100) freshly supplemented with protease inhibitors. Lysates were sheared by sonication (3 x 5 s, amplitude 2) and cleared by centrifugation at 14,000 rcf for 10 minutes. Biotinylated proteins were enriched by incubating with Streptavidin magnetic beads (Pierce, ThermoFisher) for 2 hours at 4°C. Beads were then transferred to the KingFisher system (ThermoFisher) and washed 2 times with RIPA-buffer, 2 times with Tris-buffer (50 mM Tris, pH 7.5, 150 mM NaCl) and one time with 1M KCl, with 100 mM Na2CO3, and with 2 M urea. MS sample preparations were performed as described above with the PAC procedure employing proteolytic digestion on the streptavidin beads for 1 hour.PrideMS analysis Resulting peptides were purified and concentrated on C18 stage tips before being subjected to LCMS analysis with an Easy nanoLC system coupled directly to a Thermo Scientific Orbitrap Exploris 480 mass spectrometer. MS data were acquired by Data Dependent Acquisition (DDA) and searched against the reviewed sequences from the Uniprot mouse proteome database using MaxQuant, with "match between runs" and "Label-free quantification" enabled. The MaxQuant protein group output was analyzed with Perseus or PerseusR.ProteomicsJens S. AndersenUniversity of Southern Denmark, Department of Biochemistry and Molecular Biology, Campusvej 55, 5230 Odense M, DenmarkPARTIALMus Musculus (mouse)jens.andersen@bmb.sdu.dk39515294 Imamura K, Garland W, Schmid M, Jakobsen L, Sato K, Rouvière JO, Jakobsen KP, Burlacu E, Lopez ML, Lykke-Andersen S, Andersen JS, Jensen TH. A functional connection between the Microprocessor and a variant NEXT complex. Mol Cell. 2024 84(21):4158-4174.e6 10.1016/j.molcel.2024.10.015University of Southern Denmark, Department og Biochemistry and Molecular BiologyDenmarkIn mammalian cells, primary miRNAs are cleaved at their hairpin structures by the Microprocessor complex, whose core is composed of DROSHA and DGCR8. Here, we show that 5' flanking regions, resulting from Microprocessor cleavage, are targeted by the RNA exosome in mouse embryonic stem cells (mESCs). This is facilitated by a physical link between DGCR8 and the nuclear exosome targeting (NEXT) component ZCCHC8. Surprisingly, however, both biochemical and mutagenesis studies demonstrate that a variant NEXT complex, containing the RNA helicase MTR4 but devoid of the RNA-binding protein RBM7, is the active entity. This Microprocessor-NEXT variant also targets stem-loop-containing RNAs expressed from other genomic regions, such as enhancers. By contrast, Microprocessor does not contribute to the turnover of less structured NEXT substrates. Our results therefore demonstrate that MTR4-ZCCHC8 can link to either RBM7 or DGCR8/DROSHA to target different RNA substrates depending on their structural context.A functional connection between the Microprocessor and a variant NEXT complex.Imamura Katsutoshi K, Garland William W, Schmid Manfred M, Jakobsen Lis L, Sato Kengo K, Rouvière Jérôme O JO, Jakobsen Kristoffer Pors KP, Burlacu Elena E, Lopez Marta Loureiro ML, Lykke-Andersen Søren S, Andersen Jens S JS, Jensen Torben Heick THfalseA functional connection between the Microprocessor and a variant NEXT complexIn mammalian cells, primary miRNAs are cleaved at their hairpin structures by the Microprocessor complex, whose core is composed of DROSHA and DGCR8. Here, we show that 5’flanking regions, resulting from Microprocessor cleavage, are targeted by the RNA exosome in mouse embryonic stem cells (mESCs). This is facilitated by a physical link between DGCR8 and the nuclear exosome targeting (NEXT) component ZCCHC8. Surprisingly, however, both biochemical and mutagenesis studies demonstrate that a variant NEXT complex, containing the RNA helicase MTR4 but devoid of the RNA-binding protein RBM7, is the active entity. This Microprocessor-NEXT variant also targets stem-loop containing RNAs expressed from other genomic regions, such as enhancers. In contrast, Microprocessor does not contribute to the turnover of less structured NEXT substrates. Our results therefore demonstrate that MTR4-ZCCHC8 can link to either RBM7 or DGCR8/DROSHA to target different RNA substrates depending on their structural context.2026-05-222024-07-12PXD053925NEWT:6945NEWT:3555NEWT:241368NEWT:2NEWT:157546NEWT:42528NEWT:190802NEWT:35554NEWT:9778NEWT:150475NEWT:38942NEWT:9417NEWT:7111NEWT:347515NEWT:1216979NEWT:307972NEWT:32046NEWT:544496NEWT:5180NEWT:256737NEWT:2042546NEWT:115104NEWT:1081927NEWT:1238993NEWT:67825NEWT:185579NEWT:43179NEWT:13076NEWT:4513NEWT:1249668NEWT:317NEWT:55153NCBITaxon:10407NEWT:1736309NEWT:7227NEWT:7469NEWT:885318NEWT:1211601NEWT:415540NEWT:4081NEWT:876138NEWT:554NEWT:98334NEWT:426428NEWT:237561NEWT:6928NEWT:10036NEWT:7574NEWT:1351NEWT:7215NEWT:2649997NEWT:575412NEWT:29204NEWT:272563NEWT:507601NCBITaxon:79857NCBITaxon:6157NEWT:95648NEWT:3885NEWT:746360NEWT:6239NEWT:3888NEWT:1589NEWT:470150NEWT:135622NEWT:216257NEWT:6915NEWT:9986NEWT:101510NEWT:4054NEWT:3880NEWT:272559NEWT:226186NEWT:1268063NEWT:3641NEWT:383379NEWT:8782NEWT:1263854NEWT:10029NEWT:1000589NEWT:435590NEWT:1902NEWT:85962NEWT:160488NEWT:28104NEWT:317447NEWT:7955NCBITaxon:2NEWT:985076NEWT:7959NEWT:1215323NEWT:2261NEWT:4565NEWT:1264690NEWT:515619NEWT:6192NEWT:28532NCBITaxon:38727NEWT:192875NEWT:34305NEWT:59729NCBITaxon:183674NEWT:224308NEWT:626528NEWT:216599NEWT:139927NEWT:4558NEWT:209285NEWT:216595NEWT:243230NEWT:8355NEWT:1283NEWT:931281NEWT:4550NEWT:1000561NEWT:7029NEWT:1283300NEWT:6183NEWT:6063NEWT:334747NEWT:61235NCBITaxon:79824NEWT:4787NCBITaxon:4563NEWT:5755NEWT:3218NEWT:5759NEWT:1736231NEWT:436486NEWT:6287NEWT:2242NEWT:300641NEWT:4784NEWT:727NEWT:9796NEWT:11320NEWT:725NEWT:360106NEWT:260707NEWT:287NEWT:10117NEWT:10239NCBITaxon:6191NEWT:10116NEWT:1280NEWT:1836NEWT:1735272NEWT:83334NEWT:185431NEWT:83332NEWT:29760NEWT:260704NEWT:703612NEWT:260705NEWT:80863NEWT:44685NEWT:2697049NEWT:1148NEWT:580240NEWT:11676NEWT:55571NEWT:100226NCBITaxon:6073NEWT:884204NEWT:4530NEWT:4896NEWT:6279NEWT:1123869NEWT:7370NEWT:75058NEWT:83906NEWT:607699NEWT:6282NEWT:1094343NEWT:208964NEWT:1134506NEWT:575584NEWT:296543NEWT:1773NEWT:38783NEWT:8727NEWT:1895NEWT:4006NEWT:1182590NEWT:8726NEWT:6426NEWT:6669NEWT:10090NEWT:935293NEWT:64152NEWT:749200NEWT:4120NEWT:51515NEWT:5693NEWT:8724NEWT:51511NEWT:92867NEWT:8723NEWT:990346NEWT:5334NEWT:145953NEWT:257309NEWT:100816NEWT:263NEWT:230741NEWT:284812NCBITaxon:10359NCBITaxon:1313NEWT:43330NEWT:242619NEWT:44544NEWT:632957NEWT:373995NEWT:5689NEWT:645463NEWT:544404NEWT:3702NEWT:129249NEWT:9925NEWT:8839NEWT:4232NEWT:990119NEWT:2758385NEWT:4113NEWT:837NEWT:11298NEWT:171101NEWT:714NEWT:421932NEWT:196627NEWT:408172NEWT:5691NEWT:408170NEWT:493760NEWT:260710NEWT:627025NEWT:257313NEWT:400772NEWT:1097677NEWT:3708NEWT:128161NEWT:332648NEWT:106592NEWT:536231NEWT:61674NEWT:1117957NEWT:9913NEWT:1432138NEWT:10312NEWT:1424507NCBITaxon:1773NEWT:4100NEWT:1076NEWT:6763NEWT:803NEWT:8030NEWT:29722NEWT:380394NEWT:1692259NEWT:1639NEWT:188229NEWT:3818NEWT:480NEWT:4909NEWT:180066NEWT:67767NEWT:46835NEWT:135588NEWT:376686NEWT:1843183NEWT:95486NEWT:58002NEWT:9103NEWT:4577NEWT:1416333NEWT:5664NEWT:2157NEWT:418985NEWT:146479NEWT:10306NCBITaxon:2759NEWT:1911079NEWT:8022NEWT:145943NCBITaxon:4932NEWT:595536NEWT:593117NEWT:3635NEWT:5811NEWT:235443NEWT:1480154NEWT:1274414NEWT:27606NEWT:59202NEWT:9975NEWT:38865NEWT:3197NEWT:9615NEWT:10299NEWT:860688NEWT:411483NEWT:884019NEWT:198215NEWT:411490NEWT:983964NEWT:169963NEWT:36329NEWT:1147787NCBITaxon:3044782NEWT:499175NEWT:72407NEWT:476272NEWT:349741NEWT:9606NEWT:367830NEWT:157295NEWT:641501NEWT:178616NEWT:410289NEWT:373153NEWT:7668NEWT:915099NEWT:74940NEWT:9721NEWT:1450511NEWT:360094NEWT:470NEWT:411901NEWT:1313NEWT:411469NEWT:84023NEWT:9838NEWT:105884NCBITaxon:9615NCBITaxon:5811NEWT:58334NEWT:411464NEWT:1193501NEWT:3055NEWT:6326NEWT:6689NEWT:411460NEWT:2762NEWT:5476NEWT:1174673NEWT:562NEWT:411470NEWT:33952NEWT:2094720NCBITaxon:2697049NEWT:1274432NEWT:1274426NEWT:1423NEWT:4932NEWT:70448NEWT:9825NEWT:1274423NEWT:3603NEWT:393765NEWT:698936NEWT:2759NEWT:3847NEWT:39946NEWT:9823NEWT:178876NEWT:9940NEWT:327160NEWT:573NEWT:521001NEWT:9031NEWT:1274420NEWT:7091NEWT:578458395152940000-0002-6091-140X