Liquid-like condensates have been thought to be sphere-like. Recently, various condensates with filamentous morphology have been observed in cells. One such condensate is the TIS granule network that shares a large surface area with the rough endoplasmic reticulum and is important for membrane protein trafficking. It has been unclear how condensates with mesh-like shapes but dynamic protein components are formed. In vitro and in vivo reconstitution experiments revealed that the minimal components are a multivalent RNA-binding protein that concentrates RNAs that are able to form extensive intermolecular mRNA–mRNA interactions. mRNAs with large unstructured regions have a high propensity to form a pervasive intermolecular interaction network that acts as condensate skeleton. The underlying RNA matrix prevents full fusion of spherical liquid-like condensates, thus driving the formation of irregularly shaped membraneless organelles. The resulting large surface area may promote interactions at the condensate surface and at the interface with other organelles. Despite lacking a surrounding lipid membrane, membraneless organelles are micron-sized structures that compartmentalize the subcellular space to organize biological reactions . Most knTIS granules are formed through assembly of the RNA-binding protein TIS11B. TIS granules have tubule-like structures and generate a reticular meshwork that is intertwined with the rough endoplasmic reticulum (ER), one of the major sites of protein translation. The mesh-like morphology of the TIS granule network allows it to share a lot of surface area with the ER, thus, generating a large condensate–organelle interface. TIS granules are present in various cell types under physiological conditions and provide a translation environment for mRNAs that contain several AU-rich elements in their 3′ untranslated regions (3′UTRs). Translation in the TIS granule–ER interface, the so-called TIGER domain, allows membrane proteins to form specific protein complexes, indicating that TIS granules are important for the trafficking of plasma membrane proteins .Xenopus oocyte maturation, the ER and maternal mRNAs localize to the vegetal pole, which is critical for proper embryonic patterning . When we introduced different point mutations to disrupt RNA binding  of FUS . ExpressImportantly, both FUS-TIS and SUMO-SIM-TIS show fast fluorescence recovery in FRAP experiments , indicatFUS 3′UTR did not change the morphology of the sphere-like condensates formed by FUS-TIS  or ELAVL1 (HuR) (We recombinantly expressed and purified FUS-TIS . FUS-TIS FUS-TIS . HoweverL1 (HuR) , we obseL1 (HuR) . ImportaL1 (HuR) .All phase separation experiments were performed at two time points (after 2 and 16 hr of incubation) in the presence of 5% dextran and at RNA concentrations spanning three orders of magnitude. Network formation was already observed at the early time point, but longer incubation led to formation of a more extensive network  and 2A–DThe three network-forming RNAs are longer than the sphere-forming RNA . To examTo identify the responsible determinants for network formation, we focused on the 18 3′UTRs that were shorter than 2000 nt and correlated their ability for network formation with several parameters. Within this size-restricted cohort, the number of AU-rich elements or the GC-content of the RNA had no influence on network formation . We thenCan we use RNA-fold-based structure prediction as a tool to identify network-forming RNAs? Ensemble diversity is the number of potential RNA structures that are predicted for a given RNA . As enseTNFSF11 3′UTR that contains several LDRs  of the tested RNAs were predicted correctly with respect to their sphere- or network-forming abilities . As the ral LDRs  and intrral LDRs . The TNFormation . Taken tCD47 and ELAVL1 3′UTRs and corresponds to 27 and 32 ng/µl, respectively  that are unable to induce network formation (TLR8 and MYC 3′UTRs. (D1) and from HIV (D2)  .TLR8 and MYC) generates sphere-like condensates, whereas the addition of RNAs capable of forming a crosslinked RNA network (D1-TLR8-D2 and D1-MYC-D2), which we call an RNA matrix, induces formation of mesh-like FUS-TIS condensates (A phase separation experiment with FUS-TIS confirmed that the addition of the two predominantly structured 3′UTRs (densates . Taken tTLR8 and MYC 3′UTRs with and without dimerization elements) together with SUMO-SIM as protein component. SUMO-SIM is especially suitable for this approach as all SUMO-SIM condensates are sphere-like. Our goal was to recruit mRNAs that are able to form a pervasive RNA interaction network into SUMO-SIM condensates using the MS2 system , we observed mesh-like condensate formation in vivo, indicating that we are able to reconstitute mesh-like condensates in living cells , provided by Calvin H. Jan. Cells were maintained at 37°C with 5% COAll primers are reported in Point mutations were generated using QuikChange Lightning Multi Site-Directed Mutagenesis Kit  if not otherwise stated. pcDNA-puro-mGFP  was generated from pcDNA-puro-eGFP using the primer eGFP A207K. For TIS11B RBD mutants, the following primers were used: TIS11B C135H, TIS11B C173H, TIS11B F137N, TIS11B F175N, TIS11B K116L, TIS11B K154L, TIS11B R116L, and TIS11B K152L. We called the RBD mutants CC, FF, KK, and RK because the mutated amino acids are C135H/C173H, F137N/F175N, K116L/K154L, and R114L/K152L, respectively.HindIII and ApaI; Fragment 3 was digested with ApaI and EcoRI. To generate full-length TIS-HuR chimera, Fragment 1–2 and Fragment 3 were cloned into the pcDNA3.1-puro-mCherry vector with HindIII and EcoRI restriction sites.TIS-HuR RBD contains the N-terminus of TIS11B  fused to RRM1/2 of HuR (aa 19–189) fused to the C-terminus of TIS11B (TIS11B C); (aa 182–338). This construct was generated using PCR amplification of three overlapping fragments. Fragment 1: TIS11B N was PCR-amplified from the mCherry-TIS11B construct with primers TIS-HuR 1F and TIS-HuR 1R. Fragment 2: RRM1/2 of HuR was PCR-amplified from the eGFP-HuR-3′UTR construct with primers TIS-HuR 2F and TIS-HuR 2R. Fragment 3: TIS11B C was PCR-amplified from the mCherry-TIS11B construct with primers TIS-HuR 3F and TIS-HuR 3R. A ligation PCR was performed to ligate Fragment 1 and Fragment 2 to generate Fragment 1–2 with primers TIS-HuR 1F and TIS-HuR 2 R-2. Then Fragment 1–2 was digested with AgeI and BsrGI restriction sites.The pmCherry-SUMO10-SIM5 construct was a gift from the lab of Liam J. Holt (NYU). To generate the SUMO-SIM-TIS (TIS11B RBD fused to the N-terminus of SUMO10-SIM5) fusion protein, two overlapping fragments were PCR-amplified. Fragment 1, mCherry, was PCR-amplified from the mCherry-TIS11B construct with primers TIS-SUMO-SIM 1F and TIS-SUMO-SIM 1R. Fragment 2, the RBD (aa 114–181) of TIS11B, was PCR-amplified from the mCherry-TIS11B construct with primers TIS-SUMO-SIM 2F and TIS-SUMO-SIM 2R. A ligation PCR was performed to generate mCherry-TIS11B RBD with primers SUMO-SIM 1F and TIS-SUMO-SIM 2R. mCherry-TIS11B RBD was cloned into the SUMO10-SIM5 construct with BsrGI and EcoRI restriction sites.For the FUS-TIS (FUS IDR fused to the N-terminus of TIS11B RBD) fusion protein, two overlapping fragments were PCR-amplified. Fragment 1: the IDR (aa 1–214) of FUS was PCR-amplified from the pcDNA-puro-eGFP-FUS-3′UTR vector with primers FUS-TIS 1F and FUS-TIS 1R. Fragment 2: the RBD (aa 114–181) of TIS11B was PCR-amplified with primers FUS-TIS 2F and FUS-TIS 2R. A final ligation PCR was performed to ligate two PCR fragments to the full-length FUS-TIS chimera with primers FUS-TIS 1F and FUS-TIS 2R. The full-length FUS-TIS was cloned into pcDNA3.1-puro-mGFP vector with BsrGI and EcoRI restriction sites and cloned into the pcDNA3.1-puro-BFP vector with the same restriction sites.For the pcDNA3.1-puro-BFP-FUS-TIS construct, a nuclear export signal (nes) was added upstream of the FUS IDR to increase cytoplasmic localization. It was obtained from pcDNA3.1-puro-mCherry-nes-FUS-TIS  using HindIII and EcoRI restriction sites.The RBD of HuR (aa 19–189) was PCR-amplified from the eGFP-HuR-3′UTR construct with primers HuR RBD F and FUS-HuR 2R. HuR RBD was cloned into pcDNA3.1-puro-mGFP vector with HindIII and EcoRI restriction sites.To generate the FUS-HuR RBD (FUS IDR fused to the N-terminus of HuR RBD) fusion protein, two overlapping fragments were PCR-amplified. Fragment 1: the IDR (aa 1–214) of FUS was PCR-amplified from the pcDNA-puro-eGFP-FUS-3′UTR vector with primers FUS-HuR 1F and FUS-HuR 1R. Fragment 2: the RBD (aa 19–189) of HuR was PCR-amplified from the eGFP-HuR-3′UTR construct with primers FUS-HuR 2F and FUS-HuR 2R. A final ligation PCR was performed to ligate two PCR fragments to obtain FUS-HuR chimera with primers FUS-HuR 1F and FUS-HuR 2R. The full-length FUS-TIS was cloned into pcDNA3.1-puro-mGFP vector with TLR8 construct, the TLR8 3ʹUTR was PCR-amplified from HeLa genomic DNA with primers TLR8-MS2 F and TLR8-MS2 R. The TLR8 3ʹUTR was cloned into the pcDNA-puro-eGFP vector with BsrGI and EcoRI restriction sites.Contructs for in vivo reconstitution. To generate the eGFP-3ʹUTR of MYC construct, the MYC 3ʹUTR was PCR-amplified from HeLa genomic DNA with primers MYC-MS2 F and MYC-MS2 R. The MYC 3ʹUTR was cloned into the pcDNA-puro-eGFP vector with BsrGI and EcoRI restriction sites.To generate the eGFP-3ʹUTR of D1-3ʹUTR of TLR8-D2 construct (TLR8 3ʹUTR with RNA dimerization elements D1 and D2), the D1-TLR8 3ʹUTR-D2 was PCR-amplified from the TLR8 3ʹUTR PCR product with primers D1D2-MS2 F1 and D1D2-MS2 R. D1-TLR8 3ʹUTR-D2 was cloned into the pcDNA-puro-eGFP vector with BsrGI and EcoRI restriction sites.To generate the eGFP-D1-3ʹUTR of MYC-D2 construct (MYC 3ʹUTR with RNA dimerization elements D1 and D2), the D1-MYC 3ʹUTR-D2 was PCR-amplified from the MYC 3ʹUTR PCR product with primers D1D2-MS2 F2 and D1D2-MS2 R. The D1-MYC 3ʹUTR-D2 was cloned into the pcDNA-puro-eGFP vector with BsrGI and EcoRI restriction sites.To generate the eGFP-EcoRI and XhoI restriction sites.For the eGFP-6xMS2 binding site construct, 6xMS2 binding site was cloned into pcDNA-puro-eGFP vector with TLR8-6xMS2 construct, the TLR8 3ʹUTR was PCR-amplified from HeLa genomic DNA with primers TLR8-MS2 F and TLR8-MS2 R. The TLR8 3ʹUTR was cloned into the pcDNA-puro-eGFP-6xMS2 vector with BsrGI and EcoRI restriction sites.To generate the eGFP-3ʹUTR of MYC-6xMS2 construct, the MYC 3ʹUTR was PCR-amplified from HeLa genomic DNA with primers MYC-MS2 F and MYC-MS2 R. The MYC 3ʹUTR was cloned into the pcDNA-puro-eGFP-6xMS2 vector with BsrGI and EcoRI restriction sites.To generate the eGFP-3ʹUTR of D1-3ʹUTR of TLR8-D2-6xMS2 construct (TLR8 3ʹUTR with RNA dimerization elements D1 and D2), the D1-TLR8 3ʹUTR-D2 was PCR-amplified from the TLR8 3ʹUTR PCR product with primers D1D2-MS2 F1 and D1D2-MS2 R. The D1-TLR8 3ʹUTR-D2 was cloned into the pcDNA-puro-eGFP-6xMS2 vector with BsrGI and EcoRI restriction sites.To generate the eGFP-D1-3ʹUTR of MYC-D2-6xMS2 construct (MYC 3ʹUTR with RNA dimerization elements D1 and D2), the D1-MYC 3ʹUTR-D2 was PCR-amplified from the MYC 3ʹUTR PCR product with primers D1D2-MS2 F2 and D1D2-MS2 R. The D1-MYC 3ʹUTR-D2 was cloned into the pcDNA-puro-eGFP-6xMS2 vector with BsrGI and EcoRI restriction sites.To generate the eGFP-AgeI and BsrGI to release the mCherry fragment. The MS2-mCherry was cloned into the digested pmCherry-SUMO10-SIM5 vector with AgeI and BsrGI restriction sites.To generate the MS2-mCherry-SUMO10-SIM5 (MS2 coat protein fused to the N-terminus of mCherry-SUMO10-SIM5), MS2-mCherry was PCR-amplified from the pcDNA-MS2-mCherry-HuR vector  with priLipofectamine 2000 (Invitrogen) was used for all transfections.TNFα ARE-1 (AU-rich element) was purchased from Dharmacon. mCherry-tagged constructs were transfected into HeLa cells with or without 3′-biotinylated RNA oligonucleotides. Twenty-four hours after transfection, HeLa cells were lysed with 200 µl ice-cold NP-40 lysis buffer  for 30 min. Then, cell lysates were spun down at 20,000 g for 10 min at 4°C. The supernatant was transferred to a pre-cooled tube and diluted with 300 µl ice-cold dilution buffer . Streptavidin C1 beads (Invitrogen) were added to each tube and rotated for 1 hr at 4°C. Beads were washed three times with wash buffer . Lastly, 2× Laemmli sample buffer was added to the beads, boiled at 95°C for 10 min, and cooled on ice before loading on SDS page gels. This was followed by western blotting.To examine the RNA-binding activity of TIS11B WT and TIS11B RBD mutants, RNA oligonucleotide pulldown was performed as described previously . A 3′-biAB_477593), mouse anti-mCherry , rabbit anti-HuR , IRDye 680RD donkey anti-rabbit IgG secondary antibody , and IRDye 800CW donkey anti-mouse IgG secondary antibody .Western blots were performed as described previously . ImagingXbaI and EcoRI restriction sites. mGFP-FUS-TIS-Strep-tag II was PCR-amplified from pcDNA-mGFP-FUS-TIS construct with primers mGFP F and TIS RBD-Strep-tag R. The Strep-tag II sequence was incorporated into primer TIS RBD-Strep-tag R. Full-length mGFP-FUS-TIS-Strep-tag II was cloned into pET28a-6xHis-MBP backbone with NheI and EcoRI restriction sites.mGFP-FUS-TIS was cloned into the bacterial expression vector pET28a, which was a gift from the lab of Dirk Remus (MSKCC). At the N-terminus of mGFP, we added a 6xHis-MBP tag, followed by a Tev protease cleavage site. At the C-terminus of TIS11B, we added a Strep-Tag II (SAWSHPQFEK). The 6xHis-MBP tag was PCR-amplified from pDZ2087 construct  with primers MBP F and MBP R. Full-length 6xHis-MBP was cloned into pET28a backbone with Escherichia coli (New England Biolabs). Two fresh colonies were cultivated overnight in 2 × 50 ml SOB medium at 37°C, and then 4 × 25 ml bacteria were transferred to 4 × 1 liter SOB medium to grow at 37°C until OD600 reached 0.6. Bacteria were then kept in a 4°C cold room until 18:00. Protein expression was induced by addition of 1 mM IPTG, and bacteria were incubated at 16°C overnight.To purify high-quality FUS-TIS protein, we used three steps of purification. Step 1: His-Ni purification; step 2: Strep-Tag II purification; and step 3: size exclusion chromatography. pET28a-6xHis-MBP-Tev cleavage site-mGFP-FUS-TIS-Strep-tag II was transformed into BL21 g for 10 min, and the pellet was resuspended in 100 ml cold lysis buffer. High-salt lysis buffer  was used to remove nucleic acid contamination. Bacteria were sonicated on ice for 60 min with on/off interval of 1 and 2 s. The lysate was centrifuged at 13,000 g for 30 min.Bacteria were centrifuged at 6000 A 6 ml Ni-NTA (Qiagen) was washed with five column volumes of wash buffer 1 . After centrifugation, the supernatant of the bacteria lysate was transferred into new 50 ml Falcon tubes and incubated with Ni-NTA (Qiagen) at 4°C for 30 min. Then, the sample was transferred into three gravity columns and washed respectively with 40 ml wash buffer 2 , followed with 10 ml wash buffer 3 . Then, the sample was eluted with 30 ml elution buffer 1 .After Ni-NTA purification, the eluted sample was transferred to a 5 ml StrepTrap column , which was pre-equilibrated with elution buffer 2  using the AKTA Purifier system . The target protein was eluted with 20 ml elution buffer 3 .The eluted protein was concentrated using Amicon Ultra-centrifugal filters-50K (Millipore). The concentrated sample was further purified by gel filtration on HiLoad 16/600 Superdex200 column  in elution buffer 2  using the AKTA Purifier system .The fractions representing the monomeric protein were collected and concentrated with Amicon Ultra-centrifugal filters-50K (Millipore). The quality of the final protein product was examined by SDS PAGE. The OD260/280 ratio of the final protein product was 0.52, measured with NanoDrop. The protein concentration was measured by Bradford assay (Bio-Rad). The protein was aliquoted into PCR tubes and flash-frozen in liquid nitrogen and then stored at −80°C.TAATACGACTCACTATAGGG) was incorporated into the forward primers used to amplify the DNA templates. After in vitro transcription, products were DNase-treated and run on agarose gels to evaluate the integrity and size of the RNA.All RNAs were in vitro transcribed using the T7 MEGAscript kit (Ambion by Life Technologies). All DNA templates used for in vitro transcription were PCR-amplified and purified with a gel extraction kit (Qiagen). The T7 promoter  were generated as follows. For D1a-In vitro transcription was performed in a 20 µl volume according to the manufacturer’s guidelines. To generate Cy3- or Cy5-labeled RNA, 0.2 µl of 2.5 mM Cy3-UTP or Cy5-UTP (Enzo Life Sciences) was added into the in vitro transcription reaction.The transcription reaction was incubated 3 hr at 37°C in a PCR machine. All transcribed RNAs were digested with DNase for 30 min at 37°C, then precipitated with LiCl for 4 hr to overnight at −20°C. RNAs were centrifuged at 13,000 for 15 min, and the RNA pellets were washed with 70% ethanol three times. RNAs were dissolved in nuclease-free water and stored at −20°C. The concentration of RNAs was measured by NanoDrop One.2, 25 mM Tris-Cl, pH 7.4, 1 mM DTT, 2.5% glycerol, 5% dextran T500 [Pharmacosmos]). Without 5% dextran, there is no phase separation of FUS-TIS protein at the concentration of 10 µM. ZnCl2 was added as the RBD of TIS11B has two zinc finger motifs. Only in the phase separation assay shown in To allow phase separation, purified 6xHis-MBP-mGFP-FUS-TIS-Strep tag II protein stock was incubated with Tev protease for 1 hr at room temperature (RT) to cleave off the 6xHis-MBP tag. mGFP and Strep tag II were not cleaved off. All phase separation assays were performed in 20 µl phase separation buffer  was then transferred into a 384-well glass-bottom microplate (Greiner Bio-One). The chambers of the microplate were pre-treated with 1 mg/ml bovine serum albumin (BSA) (NEB) for 30 min before aspirating the BSA. The microplate was kept in the dark at RT for 2 or 16 hr, followed by imaging of the condensates using confocal microscopy. All phase separation experiments were performed at least three times.After 1 hr of Tev protease digestion, the FUS-TIS protein stock was diluted into the desired concentrations with protein stock buffer  and centrifuged at 13,000 For RNA-only phase separation experiments, Cy5-labeled RNA was diluted with the same phase separation buffer into desired concentrations in a 20 µl volume. The mixture (20 µl) was then transferred into a 384-well glass-bottom microplate. The chambers of the microplate were pre-treated with 1 mg/ml BSA for 30 min before aspirating the BSA. The microplate was kept in the dark at RT for 16 hr, followed by imaging of the Cy5-labeled RNA using confocal microscopy.2. Images were prepared with the commercial ZEN software black edition (Zeiss).Confocal imaging was performed using ZEISS LSM 880 with Airyscan super-resolution mode. Z stack images were captured with an interval size of 487 nm. A Plan-Apochromat 63x/1.4 Oil objective (Zeiss) was used. For live-cell imaging, HeLa cells were plated on 3.5 cm glass-bottom dishes (Cellvis) and transfected with the indicated constructs. Fourteen hours after transfection, cells were imaged in cell culture medium while incubating in a LiveCell imaging chamber (Zeiss) at 37°C and 5% COThe morphology of the condensates was scored by two independent scientists who agreed on the classification. To examine the ability of a specific RNA to induce network formation, several RNA concentrations were tested, for instance, for RNAs with a length of ~1000 nt, we tested concentrations from 50 nM (~16 ng/µl) to 750 nM (~250 ng/µl). If an RNA induced network formation within the concentration range, we considered it as network-forming RNA. If an RNA did not induce network formation even at 750 nM concentration, it was considered as a sphere-forming RNA.FRAP experiments were performed with a ZEISS LSM 880 confocal microscope. A Plan-Apochromat 63x/1.4 Oil objective (Zeiss) was used.For FRAP of phase separation experiments, 10 µM mGFP-FUS-TIS was mixed with specific RNAs to induce condensate formation. Two hours after mixing, an area of diameter = 1 µm was bleached with a 405 nm and 633 nm laser. GFP or Cy5 (in the case of RNA) fluorescence signal was collected over time.2. An area of diameter = 1 µm was bleached with a 405 nm laser. GFP or mCherry fluorescence signal was collected over time. For FRAP of TIS granules at 5 hr after transfection, GFP fluorescence signal was only collected for 10 s after bleaching as the TIS granules are highly mobile.For FRAP in live cells, HeLa cells were plated on 3.5 cm glass-bottom dishes (Cellvis) and transfected with the indicated constructs. Five or sixteen hours after transfection, cells were imaged in cell culture medium while incubating in a LiveCell imaging chamber (Zeiss) at 37°C and 5% COThe prebleached fluorescence intensity was normalized to 1, and the signal after bleaching was normalized to the prebleach level.Custom Stellaris EGFP FISH probes were described previously . RNA-FISIn order to examine whether specific mRNAs are enriched in the FUS-TIS granule, line profile analysis was performed. Line profiles were generated with FIJI (ImageJ). A straight line was drawn across the FUS-TIS granule, indicated by the arrows shown in the figures. Fluorescence signals along the straight line of FUS-TIS protein and the examined mRNAs were calculated with the plot profile tool in FIJI. The Pearson’s correlation coefficient (r) of two fluorescence signals was calculated with Excel.CLCA2 1.7 µg/µl, TLR8 1.6 µg/µl, HNRNPH3 1.9 µg/µl, LHFPL6 1.7 µg/µl, TNFSF11 1.8 µg/µl, TSPAN13 1.7 µg/µl). RNAs were incubated at 95°C for 2 min in a PCR machine and then incubated on ice for 2 min. RNAs were kept at 37°C for 2 hr. Also, 1 µl native agarose gel loading buffer  was added into the RNA. A total of 1 µg RNA was loaded into the 1% agarose gel made with the Tris-acetate-EDTA (TAE) buffer for electrophoresis with TAE buffer.RNA native agarose gel electrophoresis was performed as described previously with a few modifications . For sphTLR8 3′UTR, MYC 3′UTR, D1a-TLR8-D2, D1b-MYC-D2), each RNA was diluted into 4 µl buffer A  to a final concentration of 2 µM. RNAs were incubated at 95°C for 2 min in a PCR machine and then incubated on ice for 2 min. Also, 2 µl TLR8 3′UTR or 2 µl MYC 3′UTR were each diluted with 2 µl buffer A. A 2 µl TLR8 3′UTR and 2 µl MYC 3′UTR were mixed together. Then, 2 µl D1a-TLR8-D2 or 2 µl D1b-MYC-D2 were each diluted with 2 µl buffer A. Also, 2 µl D1a-TLR8-D2 and 2 µl D1b-MYC-D2 were mixed together. The final concentration of each RNA was 1 µM  in 4 µl buffer A. RNAs were kept at 37°C for 2 hr. A 1 µl native agarose gel loading buffer was added to the RNA. A total of 1 µg RNA was loaded onto the 2% agarose gel made with the Tris-borate-EDTA (TBE) buffer for electrophoresis with TBE buffer.For RNAs containing dimerization elements  the average length of an mRNA is 3500 nt, which corresponds to an average molecular weight of 1155 kDa; and (3) there are between 10 and 10,000 copies of mRNAs per cell  based on the following assumptions: (1) the volume of a HeLa cell is 2000 µmper cell .--MEA -d2 -p --infile=<RNA_sequences.fasta>--outfile=<RNA_sequences.RNAfold.summary>) (N = 28), 75% of them had NED values higher than 0.28, whereas 75% of the RNAs that induced sphere-like condensates had NED values lower than 0.265. We used these cut-offs to identify 3′UTRs with high or low NED values transcriptome-wide. The range of transcriptome-wide NED values is 0–0.44.The ensemble diversity of 3′UTR sequences was calculated using the RNAfold software  . Only 3′The 3′UTR length is the full-length 3′UTR length obtained from Refseq. For counting of AU-rich elements, we only considered the canonical sequence AUUUA. We counted the number of AU-rich elements in annotated 3′UTRs of mRNAs expressed in HeLa cells. All values are listed in PAR-CLIP data of HuR were analyzed from two datasets . ProcessFor all pair-wise comparisons, a two-sided Mann–Whitney test was performed. For comparisons containing more than two groups, a Kruskal–Wallis test was performed. The Pearson’s correlation coefficient is reported. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:The manuscript by Ma et al. investigates the filamentous condensates formed by RNA-protein interactions in cells that have been shown to be ER associated. Since most phase transition condensates are spherical and liquid droplets, the morphology of the mesh-like condensates is striking-clearly visually different. By a variety of experimental approaches, both in vitro and in vivo, the mesh-like condensate is found to be likely due to RNA with "disordered" regions that forms an interacting network of RNA strands bound by proteins with multivalent domains.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by James Manley as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "A multivalent RNA matrix drives formation of mesh-like condensates" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.In this study Ma et al. investigate the mechanisms influencing the unique morphology of TIS granules. TIS granules are elongated, filamentous condensates that form extensive contacts with the ER. The authors use a combination of in vivo and in vitro reconstitution experiments using a TIS-FUS fusion protein and various RNAs. They find that long unstructured RNAs are best at generating condensates resembling TIS granules. In contrast, RNAs with secondary structure form rounder condensates. The results are very intriguing and provocative and lend support for a key role for RNA in dictating the structure of membraneless organelles.The reviewers have numerous suggestions for improving the manuscript, almost all of them requiring changes to the text. Most of these changes recommended have to do with clarity but in some cases the reviewers felt that there should be caveats added, for instance as to the existence of the "mesh". Some conclusions should be modified and referencing expanded as suggested. Some validation experiments using mutations and control constructs with the RBD of TS11B are suggested, but are not required, however you may have this additional data or desire to include it.The authors should determine whether their title is too broad, since the mesh-like nature of the condensates is inferred.Reviewer #1:The manuscript by Ma et al. investigates the filamentous condensates formed by RNA-protein interactions in cells that have been shown to be ER associated. Since most phase transition condensates are spherical and liquid droplets, the morphology of the mesh like condensates is striking-clearly visually different. By a variety of experimental approaches, both in vitro and in vivo, the mesh like condensate is found to be likely due to RNA with "disordered" regions that forms an interacting network of RNA strands bound by proteins with multivalent domains.The definition of mRNAs with disordered regions is a relatively new concept. Although mRNA can adopt secondary structures, it is generally considered to be a floppy molecule without much intrinsic structure. The authors use a score they derive from a folding program, the NED, that can distinguish RNAs with higher structure . These programs are usually suggestive rather than predictive and it's not particularly clear exactly what constitutes a disordered region. A test of the RNAs with higher NEDs (less structured) gives an encouraging 79% correspondence with mesh formation. RNAs with low NEDs sometimes could form meshes however . The line between high and low NEDs seems therefore arbitrary, as if another factor is missing that would make it clearer what is needed for network formation. It's not evident exactly how much the protein component contributes to the mesh formation in vivo, but apparently, it's a major contributor, more than the NED would suggest.And then there's the question as to what all this means. The authors speculate about it in the Discussion . The functional consequences of mesh disruption have been partially addressed experimentally in a previous publication as aiding in the localization and insertion of membrane proteins and perhaps co-translational assembly of protein complexes. This latter possibility is the most exciting, since it is not clear what role mRNA might play in the assembly of multipolypeptide complexes. A search through the databases might yield some physiological relevance for the mRNAs capable of intermolecular hybridization. This would be a major step toward understanding its contribution to cell physiology.In all, I feel this work advances our insights into mechanisms leading to the spatial organization and compartmentalization in the cell. It is thoroughly done and at times gets into complex experimentation with a lot of variables (I had to read it four times), so will be a bit of a slog for the average reader. Nonetheless, the extra effort yielded new insights (to me at least) about the possibility of mRNA scaffolds controlling cell homeostasis.Reviewer #2:TIS granules form in various animal cells under physiological conditions. They display liquid-like properties typical of condensates that form during liquid-liquid phase separation and yet have a mesh-like morphology characteristic of condensates that undergo gelation. In their manuscript, Ma and co-authors aim to address how the mesh-like architecture of TIS granules arises in light of their liquid-likes properties. The authors propose that interacting mRNAs that enrich in TIS granules form an underlying network, which drives the irregular shape of TIS granules while allowing rapid exchange of the TIS granule resident proteins. The authors propose that the RNA structure confers the ability of mRNAs to engage in intermolecular RNA:RNA interactions, which then determines the morphology of TIS granules. The authors arrive at this model by examining the ability of in vitro transcribed 3'UTRs of 47 human mRNAs to induce the mesh-like architecture, all of which are proposed TIS11B targets. The authors find that in silico predicted unstructured 3' UTRs predominantly generated mesh-like granules, whereas, structured 3' UTRs tend to generate spherical granules. Based on these results the authors argued that unstructured RNA are more exposed and thus present more sequences amenable for intermolecular RNA:RNA base-pairing, which then drives the mesh-like granule architecture. In contrast, highly structured mRNAs cannot form such mRNA-mRNA interactions (or at least not many), preventing network formation, thus generating spherical condensates.In general, this manuscript is well written and provides a coherent message. The images are clear. The authors used in vivo and in vitro systems to address their questions. The methods are well-written and the statistics are properly analyzed. The experiment in Figure 6 A-D is strong and provides compelling evidence for the authors' proposed model. In addition, FRAP data in Figure 7F and Figure 7—figure supplement 1B demonstrated that the RNAs within in vitro reconstituted TIS granules exchange less than the TIS-FUS resident protein that forms the granule and lands further support that the RNA could instruct the shape and stability of RNA granules. These observations agree well with previous in vitro and in vivo measurements which demonstrated that RNAs rather than the granule proteins appear as stable components of a condensate. Overall, I don't think that the authors' model is wrong, however I question whether the authors fully demonstrated that intermolecular RNA:RNA interactions are the major if not the only mechanism that determines the shape and biophysical properties of TIS granules as suggested by the authors. The properties of TIS granules have mostly been examined from a protein point of view and much less from an RNA point of view and mostly in vitro and the evidence of sequence-driven intermolecular RNA:RNA interactions occurring in vivo is very weak. The evidence of interactions occurring in vitro is not sufficient to conclude that these interactions also form in vivo. Specifically, trans-compensatory mutations, which would perturb intermolecular base-pairing and RNA structure are missing. Co-localization data of wild type and mutated interacting TIS11B target mRNAs in cells are also missing. Because of this and possible alternative models that could explain the data, it is difficult to unequivocally conclude that the intermolecular RNA:RNA interactions occur in vivo and that they are responsible for the mesh-like architecture of TIS granules. I have several comments the authors should address before their manuscript is published. While the authors could address these concerns experimentally to bolster their model, I also believe that instead most of these concerns could simply involve amendments to the writing. These re-writings would include a possibility of alternative models, and reflect a more suggestive rather than definitive model of how branched TIS granules could form through intermolecular RNA:RNA interactions. My comments are as follows:eLife but should have some conclusions/claims rewritten or addressed. The title of the authors' paper is "A multivalent RNA matrix drives the formation of mesh-like condensates". However, the authors did not unequivocally demonstrate that multivalent intermolecular RNA:RNA interactions indeed occur in vivo and that they are responsible for the mesh-like architecture of TIS granules. In addition, alternative models could explain the data . The authors could address these concerns with revisions of their claims and by discussing alternative models rather than providing substantial amount of additional experimentation. I have listed the concerns the authors should address in my initial review. The authors should also define the NED value better . These are computational experiments and could be done quickly and should be included in the revised manuscript. Specifically, the authors should address if the NED values in Figure 3 are for 3' UTR or full-length RNA? Are the structures and NEDs of 3' UTRs shown in Figure 3D, E and G consistent with those 3' UTRs from the full-length RNAs? Are the mRNAs, which have unstructured 3'UTR also unstructured in full length? Finally, in Figure 3F, the authors should define the upper and lower limit of NED. Currently, it is difficult to judge whether the NEDs shown present a big or small difference. The authors should predict the NED for a very structured RNA, such as 24 MS2 repeats RNA or a tRNA as well as a linear RNA polymer, such as polyA or polyC RNA.The manuscript warrants publication in 1) Evidence of pervasive mRNA-mRNA interactions occurring in vivo is missing. Based on the models in Figures 7E and 6D, the authors define intermolecular mRNA-mRNA interactions when one mRNA base pairs with another and propose possible RNA sequences that could base pair. However, this model was never tested. The dimerization experiments using in vitro RNA oligomerization assays revealed that RNAs indeed pair with each other in vitro  but whether this also occurs in vivo is unknown. To test their model, the authors should mutate RNA sequences in TLR8 and MYC to prevent dimerization and test the effect of these mutations on dimerization in vitro and in vivo and to test the effect these sequences have on the granule morphology. The authors could achieve this by using trans-compensatory mutations and mRNA co-localization experiments in vivo  for the outline of these experiments). In addition, using FISH, the authors should demonstrate that wild type, interacting mRNAs co-localize in cells while mutated mRNA don't. This should be done with interacting TIS11B target mRNAs.2) The mesh-like architecture could be driven by a conformational change in the TIS11B protein induced by RNA-binding rather than by intermolecular RNA:RNA interactions. Specifically, the 3'UTRs of TIS11B target mRNAs CD47, ELAV and CD274 induce mesh-like structure of granules while the 3'UTR of FUS, which is not a target mRNAs of TIS11B does not . However, 3'UTR of FUS is still recruited to the granule, even though it does not appear to be a TIS11B target. Thus, binding of the RNA binding domain (RBD) of TIS11B to its true target could induce a conformational change in TIS11B, which could then induce condensation and branching. A similar conformation-based model was recently proposed for the phase separation of the stress granule protein G3BP1/2 . This becomes more problematic when the authors test additional 3'UTRs belonging to 47 different human mRNAs. These RNAs all contain multiple AU-rich elements and are considered targets of TIS11B. I had a hard time finding data in the authors' current manuscript or their previous work, which demonstrates that these 47 3'UTRs are bona fide TIS11B targets. It is therefore difficult to determine whether specific binding of TIS11B RBD to true targets induces a conformational change in the protein, which then triggers the mesh-like behavior of granules or whether granule branching is indeed achieved through dimerizing RNAs.3) The effect of RNA structure on intermolecular RNA:RNA interactions. RNA structure of 3'UTRs predicted in silico drives sphere or mesh-like appearance of TIS granules in vitro. More structured mRNAs cause spherical formation of the granules while less structured RNA tend to trigger mesh-like networks. As the authors propose, this could be due to availability of RNA sequences for intermolecular RNA:RNA interactions. They are exposed in unstructured mRNAs and hidden in structured ones. However, if I follow the point 2 I raised above, then the secondary structure could also hide RNA sequences in target mRNAs that TIS11B protein binds to regardless of how many predicted AU-rich sequences that 3'UTR might have. Conversely, less structured mRNAs could have more exposed sequences for TIS11B binding, which would influence the architecture of TIS granules. This model would also explain the lack of correlation between the number of AU elements and the morphology of granules .4) Reconstituted SUMO-SIM-TIS and FUS-TIS granules do not recapitulate the biophysical properties of TIS11B granules and full length TIS11B granules appear hydrogel-like. The data demonstrate that the tagged full-length TIS11B forms granules that display hydrogel-like rather than liquid-like properties. Specifically, using FRAP, the authors show that a transiently transfected full-length (FL) TIS11B fused with mCherry forms mesh-like condensates reminiscent of the endogenous TIS11B protein. They determine that 16h after of transfection only 20% of TIS11B-GFP exchanged within 120 second. This slow exchange is characteristic of gel-like condensates rather than predominantly liquid-like condensates. This measurement is in contrast to the authors' statement where they indicated that TIS granules are "liquid-like" and that the granule resident proteins are "highly mobile". In addition, to examine granules, the authors chose to work with fluorescently tagged chimeric FUS and SUMO-SIM proteins fussed with the RBD of TIS11B. In contrast to full-length TIS11B, these proteins form mesh-like granules that appear liquid-like; 75% of FUS-TIS exchanges in 25 seconds while close to 100% of SUMO-SIM fully exchanges in just a few seconds . These data indicate that the chimeras did not recapitulate the basic behavior of TIS granules. In addition, the mCherry-TIS11B protein has a different recovery than mGFP-TIS11B, with the latter one recovering to ~ 43% in 10s . The authors should address these discrepancies in recovery rates and whether TIS-granules are more gel-like instead of liquid-like. Finally, the rationale for switching from tagged FL TIS11B to FUS-TIS and SUMO-SIM-TIS is not clear particularly given that these chimeras do not recapitulate the behavior of FL TIS11B. It is therefore difficult to assess the effect of mRNAs on the biophysical properties of TIS granule resident proteins using a system that behaves fundamentally different than the endogenous system.Reviewer #3:In this study Ma et al. investigate the mechanisms influencing the unique morphology of TIS granules. TIS granules are elongated, filamentous condensates that form extensive contacts with the ER. The authors use a combination of in vivo and in vitro reconstitution experiments using a TIS-FUS fusion protein and various RNAs. They find that long unstructured RNAs are best at generating condensates resembling TIS granules. In contrast, RNAs with secondary structure form rounder condensates. The results are very intriguing and provocative and lend support for a key role for RNA in dictating the structure of membraneless organelles.The mechanisms underlying the filamentous structure of TIS-FUS granules remains unclear. The authors consider the possibility that the filaments may be assemblies of smaller condensates that stick rather than fuse with each other, forming elongated "beads on a string". This possibility could be tested by observing the morphology of the condensates at earlier time points. The authors may also want to refer to a recent theoretical study that explains how interactions between multivalent molecules can cause condensates to become kinetically arrested, i.e. unable to grow or fuse as the valency of molecules inside the condensates reach saturation (https://elifesciences.org/articles/56159). This theory may provide a good framework for the authors' observations.1) An important question addressed by this study is how non-spherical condensates are formed from dynamic protein components. Accordingly, the authors use FRAP experiments to conclude that TIS11 behaves dynamically in vivo. However, only 40% of TIS11 signal recovers after photobleaching . The authors should discuss whether this observation supports conventional liquid-like behavior, or whether a fraction of TIS11 in condensates may be non-dynamic. Stable assembly of TIS11 in vivo raises the possibility that TIS11 protein may contribute to mesh-like condensate assembly.2) In many cases, the authors base their conclusions on observations made with transfected/tagged TIS11 proteins. Whether these conclusions apply to the native protein is not discussed (is endogenous TIS11 dynamic?). Also the authors should refrain from making sufficiency conclusions based solely on the behavior of transfected proteins. For example, – the TIS11 RNA binding domain is sufficient to make mesh-like condensates in vivo – what is the evidence that this domain is generating a condensate and not just simply localizing to a pre-existing structure?3) The authors should include the control : GFP-FUS-Strep lacking the TIS11 RBD to ensure that the observed condensates require the TIS11 RBD.4) The authors should note clearly in the text and figures that the crowding agent dextran is included in all in vitro phase separation assays. They might also comment on the behavior of their construct in the absence of 5% dextran. Reviewer #1:The manuscript by Ma et al. investigates the filamentous condensates formed by RNA-protein interactions in cells that have been shown to be ER associated. Since most phase transition condensates are spherical and liquid droplets, the morphology of the mesh like condensates is striking-clearly visually different. By a variety of experimental approaches, both in vitro and in vivo, the mesh like condensate is found to be likely due to RNA with "disordered" regions that forms an interacting network of RNA strands bound by proteins with multivalent domains.The definition of mRNAs with disordered regions is a relatively new concept. Although mRNA can adopt secondary structures, it is generally considered to be a floppy molecule without much intrinsic structure. The authors use a score they derive from a folding program, the NED, that can distinguish RNAs with higher structure . These programs are usually suggestive rather than predictive and it's not particularly clear exactly what constitutes a disordered region. A test of the RNAs with higher NEDs (less structured) gives an encouraging 79% correspondence with mesh formation. RNAs with low NEDs sometimes could form meshes however . The line between high and low NEDs seems therefore arbitrary, as if another factor is missing that would make it clearer what is needed for network formation. It's not evident exactly how much the protein component contributes to the mesh formation in vivo, but apparently, it's a major contributor, more than the NED would suggest.We thank the reviewer for spending time to review our manuscript and for the insightful comments. For the 47 experimentally tested 3′UTRs, the NED values range from 0.18 – 0.38. Among the RNAs that induced mesh-like condensates (N = 28) 75% of them had NED values higher than 0.28, whereas 75% of the RNAs that induced sphere-like condensates had NED values lower than 0.265. We used these cut-offs to identify 3′UTRs with high or low NED values transcriptome-wide. The range of transcriptome-wide NED values is 0 – 0.44.We added the information to the text in the Results as well as to the Materials and methods.We agree that we do not know how much the protein component contributes as we only tested several multivalent domains. It is unclear if most or only a small fraction of proteins can form mesh-like condensates. But it is likely that the ability of proteins to form mesh-like condensates is not limited to a specific multivalent domain, as various multivalent proteins, when bound to RNA, including TIS11B, FXR1, FUS-IDR, SUMO-SIM and PR peptides form mesh-like condensates.And then there's the question as to what all this means. The authors speculate about it in the Discussion . The functional consequences of mesh disruption have been partially addressed experimentally in a previous publication as aiding in the localization and insertion of membrane proteins and perhaps co-translational assembly of protein complexes. This latter possibility is the most exciting, since it is not clear what role mRNA might play in the assembly of multipolypeptide complexes. A search through the databases might yield some physiological relevance for the mRNAs capable of intermolecular hybridization. This would be a major step toward understanding its contribution to cell physiology.We currently do not have direct experimental evidence to show the biological relevance of mesh-like condensates. Before our study, mesh-like condensates were largely viewed as aggregates. We hope that our study will motivate more and more people to incorporate mesh-like condensates into their thinking and into their experimental approaches.In all, I feel this work advances our insights into mechanisms leading to the spatial organization and compartmentalization in the cell. It is thoroughly done and at times gets into complex experimentation with a lot of variables (I had to read it four times), so will be a bit of a slog for the average reader. Nonetheless, the extra effort yielded new insights (to me at least) about the possibility of mRNA scaffolds controlling cell homeostasis.Reviewer #2:[…] My comments are as follows:The manuscript warrants publication in eLife but should have some conclusions/claims rewritten or addressed. The title of the authors' paper is "A multivalent RNA matrix drives the formation of mesh-like condensates". However, the authors did not unequivocally demonstrate that multivalent intermolecular RNA:RNA interactions indeed occur in vivo and that they are responsible for the mesh-like architecture of TIS granules. In addition, alternative models could explain the data . The authors could address these concerns with revisions of their claims and by discussing alternative models rather than providing substantial amount of additional experimentation. I have listed the concerns the authors should address in my initial review. The authors should also define the NED value better . These are computational experiments and could be done quickly and should be included in the revised manuscript. Specifically, the authors should address if the NED values in Figure 3 are for 3' UTR or full-length RNA? Are the structures and NEDs of 3' UTRs shown in Figure 3D, E and G consistent with those 3' UTRs from the full-length RNAs? Are the mRNAs, which have unstructured 3'UTR also unstructured in full length? Finally, in Figure 3F, the authors should define the upper and lower limit of NED. Currently, it is difficult to judge whether the NEDs shown present a big or small difference. The authors should predict the NED for a very structured RNA, such as 24 MS2 repeats RNA or a tRNA as well as a linear RNA polymer, such as polyA or polyC RNA.We thank the reviewer for spending the time to review our manuscript and for the insightful comments. We have discussed alternative models in the revised manuscript.Based on the reviewer’s suggestion, we changed the title. The new title is: in vivo reconstitution finds multivalent RNA-RNA interactions as drivers of mesh-like condensates.49). For the 47 experimentally tested 3′UTRs, the NED values range from 0.18 – 0.38. The range of transcriptome-wide NED values is 0 – 0.44 . We have added this information to the Materials and methods.NED values are for 3′UTRs. We used 3′UTRs in our study as they are not protected by ribosomes. We found a weak correlation between the NED values of 3′UTRs and NED values of the full-length mRNAs  Evidence of pervasive mRNA-mRNA interactions occurring in vivo is missing. Based on the models in Figures 7E and 6D, the authors define intermolecular mRNA-mRNA interactions when one mRNA base pairs with another and propose possible RNA sequences that could base pair. However, this model was never tested. The dimerization experiments using in vitro RNA oligomerization assays revealed that RNAs indeed pair with each other in vitro  but whether this also occurs in vivo is unknown. To test their model, the authors should mutate RNA sequences in TLR8 and MYC to prevent dimerization and test the effect of these mutations on dimerization in vitro and in vivo and to test the effect these sequences have on the granule morphology. The authors could achieve this by using trans-compensatory mutations and mRNA co-localization experiments in vivo  for the outline of these experiments). In addition, using FISH, the authors should demonstrate that wild type, interacting mRNAs co-localize in cells while mutated mRNA don't. This should be done with interacting TIS11B target mRNAs.In our in vitro and in vivo reconstitution experiment, the engineered RNAs have three degrees of valency  to enable multivalent RNA-RNA interactions. We used two well-studied dimerization elements: the HIV dimerization motif and the tracrRNA/crRNA dimerization motifs from the CRISPR system. Both dimerization motifs were reported in the literature to dimerize in vivo .To show the importance of RNA multivalency in our reconstitution experiment we disrupted the multivalent RNA-RNA interactions. To do so, we removed the dimerization elements D1 and D2. We showed both in vitro and in vivo that reduction of valency (lack of D1 and D2) leads to a dramatic decrease in the ability to induce formation of mesh-like condensates. This result supports our conclusion that multivalent RNA-RNA interactions are required to induce formation of mesh-like condensates.In our opinion, this experiment is equivalent to mutating the TLR8 or MYC mRNAs as in our manuscript, we only emphasize the requirement of multivalent RNA interactions. We do not focus on specific mRNAs or particular sequence-specific RNA-RNA interactions.2) The mesh-like architecture could be driven by a conformational change in the TIS11B protein induced by RNA-binding rather than by intermolecular RNA:RNA interactions. Specifically, the 3'UTRs of TIS11B target mRNAs CD47, ELAV and CD274 induce mesh-like structure of granules while the 3'UTR of FUS, which is not a target mRNAs of TIS11B does not . However, 3'UTR of FUS is still recruited to the granule, even though it does not appear to be a TIS11B target. Thus, binding of the RNA binding domain (RBD) of TIS11B to its true target could induce a conformational change in TIS11B, which could then induce condensation and branching. A similar conformation-based model was recently proposed for the phase separation of the stress granule protein G3BP1/2 . This becomes more problematic when the authors test additional 3'UTRs belonging to 47 different human mRNAs. These RNAs all contain multiple AU-rich elements and are considered targets of TIS11B. I had a hard time finding data in the authors' current manuscript or their previous work, which demonstrates that these 47 3'UTRs are bona fide TIS11B targets. It is therefore difficult to determine whether specific binding of TIS11B RBD to true targets induces a conformational change in the protein, which then triggers the mesh-like behavior of granules or whether granule branching is indeed achieved through dimerizing RNAs.We thank the reviewer for the insightful comment. It is possible that the formation of mesh-like morphology is driven by a conformational change in the RNA-binding protein. In our opinion, this model cannot fully explain our experimental results. This model would only work if all by chance all network-forming RNAs are true targets and all sphere-forming RNAs are not true targets despite having the similar numbers of AU-rich elements. AU-rich elements are the binding sites for TIS11B, but their presence does not prove that TIS11B actually binds. In the meantime, we obtained iCLIP data for TIS11B and found no difference in the number of iCLIP tags in sphere-forming vs network-forming RNAs.Besides, in our in vivo reconstitution experiment, we use the MS2 coat protein and SUMO-SIM, and mRNAs are recruited to the condensates by MS2 repeats. All RNAs have the same MS2 binding sites and are true targets of MCP-SUMO-SIM. But still, when the RNA dimerization motifs D1 and D2 were eliminated, the ability to induce mesh-like condensate formation is significantly reduced compared to RNAs that contain the D1 and D2 elements. Therefore, we do not think that our data are better explained by a model that infers a conformational change in the protein.For TIS granules, our transcriptome-wide analysis showed that TIS11B target mRNAs have a high propensity to form multivalent RNA-RNA interactions as they are enriched for large disordered regions and have high NED values. It is likely that multivalent RNA-RNA interactions are responsible for the mesh-like morphology of TIS granules. Nevertheless, we agree with the reviewer that we do not have direct evidence to show that multivalent RNA-RNA interactions happen in TIS granules. We cannot exclude the possibility that the mesh-like architecture of TIS granules could be driven by a conformational change in the TIS11B protein induced by RNA-binding. It is possible that TIS granules either use RNA interactions or a conformational change, or that both mechanisms contribute to its mesh-like morphology. We have added this alternative model to our manuscript.3) The effect of RNA structure on intermolecular RNA:RNA interactions. RNA structure of 3'UTRs predicted in silico drives sphere or mesh-like appearance of TIS granules in vitro. More structured mRNAs cause spherical formation of the granules while less structured RNA tend to trigger mesh-like networks. As the authors propose, this could be due to availability of RNA sequences for intermolecular RNA:RNA interactions. They are exposed in unstructured mRNAs and hidden in structured ones. However, if I follow the point 2 I raised above, then the secondary structure could also hide RNA sequences in target mRNAs that TIS11B protein binds to regardless of how many predicted AU-rich sequences that 3'UTR might have. Conversely, less structured mRNAs could have more exposed sequences for TIS11B binding, which would influence the architecture of TIS granules. This model would also explain the lack of correlation between the number of AU elements and the morphology of granules .Both groups of mRNAs have similar numbers of TIS11B binding sites identified by iCLIP, suggesting that similar numbers of binding sites are accessible. See our response to comment 2.4) Reconstituted SUMO-SIM-TIS and FUS-TIS granules do not recapitulate the biophysical properties of TIS11B granules and full length TIS11B granules appear hydrogel-like. The data demonstrate that the tagged full-length TIS11B forms granules that display hydrogel-like rather than liquid-like properties. Specifically, using FRAP, the authors show that a transiently transfected full-length (FL) TIS11B fused with mCherry forms mesh-like condensates reminiscent of the endogenous TIS11B protein. They determine that 16h after of transfection only 20% of TIS11B-GFP exchanged within 120 second. This slow exchange is characteristic of gel-like condensates rather than predominantly liquid-like condensates. This measurement is in contrast to the authors' statement where they indicated that TIS granules are "liquid-like" and that the granule resident proteins are "highly mobile". In addition, to examine granules, the authors chose to work with fluorescently tagged chimeric FUS and SUMO-SIM proteins fussed with the RBD of TIS11B. In contrast to full-length TIS11B, these proteins form mesh-like granules that appear liquid-like; 75% of FUS-TIS exchanges in 25 seconds while close to 100% of SUMO-SIM fully exchanges in just a few seconds . These data indicate that the chimeras did not recapitulate the basic behavior of TIS granules. In addition, the mCherry-TIS11B protein has a different recovery than mGFP-TIS11B, with the latter one recovering to ~ 43% in 10s . The authors should address these discrepancies in recovery rates and whether TIS-granules are more gel-like instead of liquid-like. Finally, the rationale for switching from tagged FL TIS11B to FUS-TIS and SUMO-SIM-TIS is not clear particularly given that these chimeras do not recapitulate the behavior of FL TIS11B. It is therefore difficult to assess the effect of mRNAs on the biophysical properties of TIS granule resident proteins using a system that behaves fundamentally different than the endogenous system.In our previous paper, we performed FRAP experiments after 16 hours of transfection of mCherry-TIS11B and observed that only about 20% of the fluorescent signal recovered in 120 seconds. This observation led to the conclusion that the mesh-like morphology of TIS granules is simply caused by their gel-like nature. Later, we performed FRAP experiments after 5 hours of transfection of GFP-TIS11B and observed that 43% of the fluorescent signal recovered in 10 seconds. At this time point, it is impossible to record longer as TIS granules move very fast in cells. Because of 43% recovery in 10 seconds, we did no longer consider TIS granules as gel-like and we described them in this manuscript as “somewhat dynamic”. At the 5-hour time point the morphology of TIS granules is irregular. Therefore we hypothesized that the mesh-like morphology of TIS granules cannot simply be explained by gel-like biophysical properties.We then used chimeric proteins to better understand how the irregularly shaped morphology is generated. When we saw that the mesh-like condensates formed by SUMO-SIM-TIS or FUS-TIS showed fast FRAP recovery (their fluorescence recovered to more than 75% and 90% within 30 seconds of bleaching), we became very excited. Based on the FRAP data, SUMO-SIM-TIS and FUS-TIS condensates would be considered to have liquid-like properties. However, based on the current knowledge this is a paradox as due to surface tension, liquid-like condensates should have a sphere-like morphology and should not be mesh-like.At that moment, we changed the focus of our study. Instead of studying how the mesh-like morphology of TIS granules is generated, we rather set out to investigate the paradoxical observation how liquid-like condensates can have a mesh-like morphology.In our opinion this is a more general question than the questions of what causes the mesh-like morphology of TIS granules, for which our experimental tools were limited at the time as we only had constructs in hands that poorly mimic the behavior of endogenous TIS granules. We would like to point out that this manuscript is not primarily about TIS granules, but rather about finding ways to explain how mesh-like condensates with dynamic protein components are formed. We did find one such mechanism: Multivalent RNA-RNA interactions can act as skeleton of mesh-like condensates. We are not saying that this is the only mechanism as there are likely other mechanisms that induce mesh-like condensates with dynamic protein components.We added a more extensive explanation on the rationale for the use of chimeric proteins to the text.Reviewer #3:In this study Ma et al. investigate the mechanisms influencing the unique morphology of TIS granules. TIS granules are elongated, filamentous condensates that form extensive contacts with the ER. The authors use a combination of in vivo and in vitro reconstitution experiments using a TIS-FUS fusion protein and various RNAs. They find that long unstructured RNAs are best at generating condensates resembling TIS granules. In contrast, RNAs with secondary structure form rounder condensates. The results are very intriguing and provocative and lend support for a key role for RNA in dictating the structure of membraneless organelles.The mechanisms underlying the filamentous structure of TIS-FUS granules remains unclear. The authors consider the possibility that the filaments may be assemblies of smaller condensates that stick rather than fuse with each other, forming elongated "beads on a string". This possibility could be tested by observing the morphology of the condensates at earlier time points. The authors may also want to refer to a recent theoretical study that explains how interactions between multivalent molecules can cause condensates to become kinetically arrested, i.e. unable to grow or fuse as the valency of molecules inside the condensates reach saturation (https://elifesciences.org/articles/56159). This theory may provide a good framework for the authors' observations.We thank the reviewer for spending the time to review our manuscript and for the insightful comments. We did confocal 3D time-lapse imaging of FUS-TIS condensates in vitro after 30 min of mixing with CD47 3′UTR RNA (network formation) or TLR8 3′UTR RNA (sphere formation). The results showed that smaller FUS-TIS condensates mixed with CD47 3′UTR RNA stick rather than fuse with each other. The data were shown in Figures 7A and 7B. In cells, the morphology of FUS-TIS condensates cannot be observed at an earlier time point as they move very fast when they are small. We thank the reviewer for suggesting to cite this paper. We added it to the new version of the manuscript.1) An important question addressed by this study is how non-spherical condensates are formed from dynamic protein components. Accordingly, the authors use FRAP experiments to conclude that TIS11 behaves dynamically in vivo. However, only 40% of TIS11 signal recovers after photobleaching . The authors should discuss whether this observation supports conventional liquid-like behavior, or whether a fraction of TIS11 in condensates may be non-dynamic. Stable assembly of TIS11 in vivo raises the possibility that TIS11 protein may contribute to mesh-like condensate assembly.In our previous paper, we performed FRAP experiments after 16 hours of transfection of mCherry-TIS11B and observed that only about 20% of the fluorescent signal recovered in 120 seconds. This observation led to the conclusion that the mesh-like morphology of TIS granules is simply caused by their gel-like nature. Later, we performed FRAP experiments after 5 hours of transfection of GFP-TIS11B and observed that 43% of the fluorescent signal recovered in 10 seconds. At this time point, it is impossible to record longer as TIS granules move very fast in cells. Because of 43% recovery in 10 seconds, we did no longer consider TIS granules as gel-like and we described them in this manuscript as “somewhat dynamic”. At the 5-hour time point the morphology of TIS granules is irregular. Therefore we hypothesized that the mesh-like morphology of TIS granules cannot simply be explained by gel-like biophysical properties.We then used chimeric proteins to better understand how the irregularly shaped morphology is generated. When we saw that the mesh-like condensates formed by SUMO-SIM-TIS or FUS-TIS showed fast FRAP recovery (their fluorescence recovered to more than 75% and 90% within 30 seconds of bleaching), we became very excited. Based on the FRAP data, SUMO-SIM-TIS and FUS-TIS condensates would be considered to have liquid-like properties. However, based on the current knowledge this is a paradox as due to surface tension, liquid-like condensates should have a sphere-like morphology and should not be mesh-like.At that moment, we changed the focus of our study. Instead of studying how the mesh-like morphology of TIS granules is generated, we rather set out to investigate the paradoxical observation how liquid-like condensates can have a mesh-like morphology.In our opinion this is a more general question than the questions of what causes the mesh-like morphology of TIS granules, for which our experimental tools were limited at the time as we only had constructs in hands that poorly mimic the behavior of endogenous TIS granules. We would like to point out that this manuscript is not primarily about TIS granules, but rather about finding ways to explain how mesh-like condensates with dynamic protein components are formed. We did find one such mechanism: Multivalent RNA-RNA interactions can act as skeleton of mesh-like condensates. We are not saying that this is the only mechanism as there are likely other mechanisms that induce mesh-like condensates with dynamic protein components.We added a more extensive explanation on the rationale for the use of chimeric proteins to the text.2) In many cases, the authors base their conclusions on observations made with transfected/tagged TIS11 proteins. Whether these conclusions apply to the native protein is not discussed (is endogenous TIS11 dynamic?). Also the authors should refrain from making sufficiency conclusions based solely on the behavior of transfected proteins. For example, – the TIS11 RNA binding domain is sufficient to make mesh-like condensates in vivo – what is the evidence that this domain is generating a condensate and not just simply localizing to a pre-existing structure?As pointed out in the manuscript and in the previous comment, the manuscript does not primarily focus on TIS granules as we currently do not have the tools to assess the material properties of endogenous TIS granules. However, our findings are likely valid for endogenous TIS granules because our transcriptome-wide analyses showed that the TIS11B target mRNAs have higher NED values than other mRNAs and thus, have a higher propensity to form multivalent RNA-RNA interactions. In our opinion, it is very likely that also multivalent RNA-RNA interactions are responsible for the mesh-like morphology of TIS granules.When the TIS RNA-binding domain was fused to SUMO-SIM or FUS IDR both chimeric proteins formed mesh-like condensates. However, the size and localization of SUMO-SIM-TIS condensates is very different from both FUS-TIS condensates and endogenous TIS granules. Therefore, it is unlikely that they simply localize to endogenous TIS granules. But we cannot exclude the possibility that FUS-TIS and SUMO-SIM-TIS localize to two different pre-existing structures. We have deleted the statement of sufficiency.3) The authors should include the control : GFP-FUS-Strep lacking the TIS11 RBD to ensure that the observed condensates require the TIS11 RBD.It was reported by Anthony Hyman’s lab that FUS IDR requires a very high concentration to undergo phase separation. With 10% dextran, a concentration of more than 20 µM is required to induce significant phase separation of the FUS IDR . In our experiment, we use 10 µM protein and 5% dextran. With this condition, we expect that the FUS IDR will not undergo phase separation. Therefore, we did not include the GFP-FUS IDR-Strep tag II as a control.In our in vivo reconstitution experiment , we use the MS2 coat protein (MCP) as an RNA binding domain to recruit specific RNAs. Without MCP or MS2 repeats, 100% SUMO-SIM condensates are sphere-like, which provides another line of evidence to support the point that specific RNAs need to be recruited into the condensates to induce network formation.4) The authors should note clearly in the text and figures that the crowding agent dextran is included in all in vitro phase separation assays. They might also comment on the behavior of their construct in the absence of 5% dextran.Without 5% dextran, there is no phase separation of FUS-TIS protein at the concentration of 10 µM. We added this information to the Materials and methods. We also added to the text and to the figure legend of Figure 2 that all phase separation experiments were performed in the presence of 5% Dextran.


TINCR and the identification of peptides derived from this open reading frame in the proteome of human stratum corneum. Our results demonstrate that TINCR is a protein‐coding RNA and suggest that the TINCR‐encoded protein is involved in keratinocyte cornification.Long non‐coding RNAs have been implicated in the regulation of a plethora of biological processes, yet it has been challenging to verify that they are truly not coding for proteins. Terminal differentiation‐induced non‐coding RNA (TINCR) is a 3.7‐kilobase mRNA that is highly abundant in epidermal keratinocytes prior to cornification. Here, we report the presence of an evolutionarily conserved open reading frame in  They represent a heterogeneous group of RNAs including mRNA‐like intergenic transcripts (lincRNAs), antisense transcripts of protein‐coding genes and others.TINCR RNA contains so‐called “TINCR box” motifs, which are 25 nucleotides long and were reported to mediate the interaction with “TINCR box” motifs in multiple cellular mRNAs. Furthermore, TINCR RNA reportedly binds to the staufen1 protein and subsequently stabilizes keratinocyte differentiation‐associated mRNAs.Terminal differentiation‐induced non‐coding RNA (TINCR) was identified in differentiating epidermal keratinocytes.TINCR that was deposited in the Uniprot database under the accession number A0A1B0GVN0. The Uniprot database was used as a reference for the proteomic analysis of human stratum corneum, and peptides corresponding to TINCR were identified in cornified envelopes.TINCR was first cloned from human hippocampus in the course of the National Institutes of Health, Mammalian Gene Collection project and was originally designated “Homo sapiens placenta‐specific 2 (non‐protein coding), mRNA” (GenBank accession number BC036545).2Here, we address the question as to whether TINCR is a non‐coding or a protein‐coding RNA.3https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi.We obtained amino acid sequences of peptides from a mass spectrometry (MS) analysis of human stratum corneum proteins that was reported in detail previously.4TINCR RNA is produced by transcription of the TINCR gene which is located at chromosome 19p13.3 and comprises 3 exons  and involucrin (IVL) Figure B. Like tTINCR has been conserved since the evolutionary divergence of the lineages leading to mammals and birds more than 300 million years ago.Amino acid sequence analysis showed that TINCR does not contain cysteine residues, whereas glutamine and lysine residues are present as potential sites of transglutamination Figure C. Compar5The presence of TINCR peptides in human stratum corneum and the conservation of the TINCR open reading frame through evolution indicate that TINCR encodes a protein. It is intriguing that TINCR is predicted to fold into a ubiquitin‐like domain which may facilitate specific interactions with other proteins. TINCR RNA is expressed in the skin where it is confined to differentiating keratinocytes of epidermisTINCR encodes a protein is definitely not compatible with its current designation as non‐coding RNA. Thus, we propose that TINCR should stand for Terminal differentiation‐INduced Cornification Regulator.Previously, TINCR was classified as a long non‐coding RNA and interactions of TINCR RNA with protein‐coding mRNAs, miRNAs and proteins were suggested to mediate effects of TINCR on keratinocyte differentiation. These interactions were not investigated in the present study, and therefore, binding of TINCR RNA to other molecules is not excluded. However, the fact that The authors have no conflict of interest. Click here for additional data file.


RNA editing is a post-transcriptional modification, which can provide tissue-specific functions not encoded in DNA. Adenosine-to-inosine is the predominant editing event and, along with cytosine-to-uracil changes, constitutes canonical editing. The rest is non-canonical editing. In this study, we have analysed non-canonical editing of microRNAs in the human brain. We have performed massively parallel small RNA sequencing of frontal cortex (FC) and corpus callosum (CC) pairs from nine normal individuals (post-mortem). We found 113 and 90 unique non-canonical editing events in FC and CC samples, respectively. More than 70% of events were in the miRNA seed sequence—implicating an altered set of target mRNAs and possibly resulting in a functional consequence. Up to 15% of these events were recurring and found in at least three samples, also supporting the biological relevance of such variations. Two specific sequence variations, C-to-A and G-to-U, accounted for over 80% of non-canonical miRNA editing events—and revealed preferred sequence motifs. Our study is one of the first reporting non-canonical editing in miRNAs in the human brain. Our results implicate miRNA non-canonical editing as one of the contributing factors towards transcriptomic diversity in the human brain. Biological systems thrive on diversity. At the molecular level, diversity is often contributed by variations occurring at different steps of information processing, namely DNA to RNA and RNA to protein. RNA sequences can differ from their DNA templates owing to transcriptional errors and RNA editing ,2. ProofMiRNAs usually binds to the 3′-untranslated region (3′-UTR) of mRNAs leading to downregulation of gene expression . The tarWe have performed small RNA sequencing in paired samples of frontal cortex (FC) and corpus callosum (CC) from nine individuals (post-mortem samples) to identify editing events in the mature microRNA population .We overlapped the identified miRNA variant sites with dbSNP version151 (All SNPs) followed by exome data to filter out variations at the DNA level. However, it is worthwhile to note here that these variations (genome encoded) can still be in the resulting transcript (miRNA) and bring out possible functional consequences ,55. FurtAfter filtering out these variations at the DNA level, the total number of canonical edits identified in FC were 255 (A-to-I = 211 and C-to-U = 44) and in CC were 177 (A-to-I = 136 and C-to-U = 41). In case of non-canonical miRNA editing, 175 and 158 events were detected in FC and CC, respectively . This obOut of total non-canonical events, 113 and 90 events were unique in FC and CC, respectively A,B. MoreWithin non-canonical editing events found in our study, C-to-A and G-to-U events accounted for over 80% . C-to-A p = 0.02) downstream of the edited cytosine and avoidance for guanosine on both sides (p = 0.02 and p = 0.009). We also found a lack of uracil (p = 0.04) downstream of the edited cytosine (p = 0.002) and downstream of the edited guanosine was observed  as per the manufacturer’s instructions. Libraries were prepared using Illumina’s TruSeq Small RNA Sample Prep Kit following the manufacture’s protocol. Two samples were loaded in one lane and 50-base single-end sequencing was done on an Illumina HiSeq 2000 platform. The small RNA sequencing data can be accessed in the sequence read archive (SRA ID: SRP063390).Data analysis was done using the published pipeline  with theTarget prediction for unedited and edited miRNAs was done using isoTar version 1.2 . We seleUnique non-canonical miRNA editing events combining FC and CC were used for analysis. This led to 58 C-to-A and 48 G-to-U events. One base upstream and one downstream of the edited base were fetched using an in-house script for the abovementioned edited miRNAs. Sequence preference around the edited base was visualized using WebLogo 3 . To lookThe methods followed in this paper can be seen in more detail in our previous publication .


In order to investigate RNA editing sites affecting IMF , we analyzed the transcriptome and genome sequencing data of a high- and low- groups composed of full-sib pairs pig with opposite IMF phenotypes. Finally, a total of 36 nonredundant RNA editing sites in the longissimus dorsi muscle, which may reveal the potential importance of RNA editing in IMF were identified. Four were selected as candidate sites associated with IMF. Our findings provide some new insights of RNA editing function in pig longissimus dorsi muscle.ACAT1), coatomer protein, subunit alpha (COPA), and nuclear receptor coactivator 3 (NCOA3), were selected as candidate RNA editing sites associated with IMF. One site located on the 3′ UTR region of growth hormone secretagogue receptor (GHSR) may regulate GHSR expression by affecting the interaction of miRNA and mRNA. In conclusion, we identified a total of 36 nonredundant RNA editing sites in the longissimus dorsi muscle, which may reveal the potential importance of RNA editing in IMF. Four were selected as candidate sites associated with IMF. Our findings provide some new insights of RNA editing function in pig longissimus dorsi muscle which useful for pig IMF breeding or human insulin resistances research.Intramuscular fat (IMF) is essential for improving the palatability and flavor of meat, and it is strongly associated with human insulin resistance. RNA editing is a widespread regulating event in different tissues. Here, we investigated the global RNA editing difference of two groups of pig with different IMF contents to find the potential editing sites affecting IMF. In this research, RES-Scanner and REDItools were used to identify RNA editing sites based on the whole genome and transcriptome sequencing data of the high and low groups composed of three full-sib pairs with opposite IMF phenotypes. A total of 295 RNA editing sites were investigated in the longissimus dorsi muscle, and 90.17% of these sites caused A to G conversion. After annotation, most editing sites were located in noncoding regions (including five sites located on the 3′ UTR regions). Five editing sites (including two sites that could lead to nonsynonymous amino acid changes) were located in the exons of genes. A total of 36 intergroup (high and low IMF) differential RNA editing sites were found in 33 genes. Some candidate editing sites, such as sites in acyl-coenzymeA: cholesterol acyltransferase 1 ( RNA editing is the post-transcriptional or co-transcriptional process which could make transcripts more complicated and results in potential functional consequences ,2. RNA eMedag) [FASN)) and MSTRG.426159 (459614 bp to LOC102160997) [Intramuscular fat (IMF) is highly related to insulin resistance  and is hMedag) ; lncRNAs2160997) ; miR-1302160997) ; and miR2160997) , are sigIn order to mining the potential effective RNA editing sites on porcine IMF, we first investigate RNA editing sites based on strand-specific RNA sequencing and whole-genome resequencing data of three full-sib paired 240-day-old pigs with different IMF contents. And then, we predicted the potential function of RNA editing sites by binding energy analysis and protein interaction network analysis. And finally, several candidate editing site affecting IMF content. This study not only explores the different RNA editing events between pigs with different IMF contents, but also provides new insight into the functional analysis of RNA editing sites.All animals experiment in our research were carried out under the ethics approval (No. IASCAAS-AE-09) of Animal Ethics Committee of the Institute of Animal Science, Chinese Academy of Agricultural Sciences on 7 September 2018.The longissimus dorsi muscle  were sampling from six 240-day-old Large White X Min pig F2 individuals  and TrSseq Nano DNA HT Sample Preparation Kit  according to the manufacturer’s recommendations. Then, the sequencing libraries were monitoring by real-time PCR on an Agilent Bioanalyzer 2100 system  for quantification and quality assessment. At last, libraries constructed above were sequenced on Illumina HiSeq  platform in Novogene Bioinformatics Technology Cooperation  by 150 bp paired-end sequencing strategy. All of the clean data have been submitted to the Genome Sequence Archive, with the accession number CRA001645.Muscle DNA and RNA sequencing libraries were generated using oligo (dT) beads by NEBNextftp://ftp.ensembl.org/pub/release-98/fasta/sus_scrofa/). In order to decrease the false positive rate, strict standards were used for RNA editing sites detection as follow: (a) DNA counts ≥10, and homozygous >0.95 (b) counts of RNA reads which differ from DNA genotype ≥3, (c) RNA editing level  ≥0.05 and false discovery rate (FDR) <0.05, and (d) candidate editing site is not located within regions of ≥3 residues and ≤6 intronic bases of a splice site.In order to strengthen robustness of detection, the RNA editing sites were detected using RES-Scanner with the main parameters —ss 1 —uniqTag 1 and default thresholds in user manual . And thehttp://www.rnanet.org/editing/main.php) and ESTs databases were randomly selected to validate the prediction accuracy of RES-Scanner. DNA and RNA were isolated from the muscle tissues of the same individuals for sequencing. The primers (All of the 295 sequences (50 bp upstream and 50 downstream of each candidate RNA editing sites) were retrieved from the reference genomes. After editing sites altering, all of the sequences were queried against the standard database of expressed sequences tags (ESTs) of swine in NCBI using BLAST algorithm. Five editing sites which could not be found in the DREP   was usedWe used bioDBnet  to conveAs described by Peng et al. , the sitFor each hyper- and hypo-editing site located at 3′UTR, we extracted the editing site and the upstream and downstream 25 base pairs  from reference genome as an unedited-type (UT) sequence and changed the editing site from A-to-G as an edited-type (ET) sequence. Then, miRANDA  was usedFor the A-to-G editing site on the exons, SIFT software  was usedTo investigate the RNA editing sites at the transcriptome-wide level in longissimus dorsi muscle, DNA sequencing and matched strand-specific RNA sequencing were performed for the six pigs. After quantification and quality filtering, a total of 542 million RNA reads were acquired from the six pigs, with an average mapping rate of 76.7%. A summary of the high-throughput sequencing result is provided in p < 0.05). As synonymous variants can disrupt transcription, splicing and mRNA stability [Consistent with the previous study in pigs, RNA editing levels of the most editing sites in muscle were low ,3,8. Mostability , we usedtability  to predip < 0.05). And, this indicated that editing events in the longissimus dorsi muscle have little chromosomal bias  [According to the in-silico blasting on NCBI, a total number of 235 (79.66%) editing sites were found covering by more than one EST clone , and a tain.php) , about 6After annotation, the RNA editing sites were located in or nearby 254 genes. We converted the ensembl number of these genes to gene official gene symbols, and then submitted genes without synonymous editing sites to Metascape for GO and interactome analysis. Together, we found that genes with editing sites participate in the function of intermuscular fat development, such as MAPK6/MAPK4 signaling. And most of genes with editing sites were associated with muscle development such as myeloid cell development, adherens junction, and skeletal system development. Moreover, genes with editing sites participate in some other biological functions such as the vascular endothelial growth factor receptor signaling pathway, the response to bronchodilators, etc. A. All thACAT1), solute carrier family 8 member A1 (SLC8A1), phosphodiesterase 3A (PDE3A), catenin alpha 3 (CTNNA3), tripartite motif containing 72 (TRIM72), and follistatin like 1 (FSTL1), with the central of growth hormone secretagogue receptor (GHSR).A total of 36 hyper- or hypo- editing sites in 33 genes were investigated in all of the editing sites. Most of the sites were also in the introns of genes, and only two sites were located in the 3′UTR region of the genes A. Among COPA), ACAT1, and nuclear receptor coactivator 3 (NCOA3).The protein–protein interaction (PPI) networks were also constructed using all of the genes containing edited sites in the longissimus dorsi muscle A. As shoZNF543). Five miRNA-mRNA interaction changes were found in the 3′ UTR region of GHSR. Seven miRNA-mRNA interaction gains were found in the 3′UTR region of GHSR  appearing in the six pigs’ longissimus dorsi muscle, and searched the alternative splicing status of all the DE genes. Finally, 23 genes have alternative splicing , but no ACAT1 and NCOA3 were found in one PPI MCODE network. In this MCODE network, ACAT1 and NCOA3 could directly interact with Ras Homolog Family Member A (RHOA), and F-Box and WD repeat domain containing 7 (FBXW7) proteins  [DDX6 plays essential roles in adipogenesis and in the alternative splicing of peroxisome proliferator activated receptor gamma (PPARG) and Lipin 1 (Lpin1) [PSMB2 can be expressed in human brown adipose tissue [HSP40 plays a vital role to obesity-induced insulin resistance and type 2 diabetes (T2D) [DNAJA1 pathway in swine [6A-dependent CDK2 expression, mediated by FTO Alpha-Ketoglutarate Dependent Dioxygenase (FTO) and YTH N6-Methyladenosine RNA Binding Protein 2 (YTHDF2), contributes to adipogenesis inhibition [COPA: g.48255C>T polymorphisms showed effects on backfat thickness. As most of the protein in this MCODE network is associated with adipogenesis, we inferred that COPA may be a candidate gene affecting IMF content. And more research, such as RNA pulldown and western blotting experiment could be performed to validate the editing events’effect on interacted proteins.COPA was also found in another PPI MCODE network containing Chaperonin Containing TCP1 Subunit 6B (CCT6B), Replication Protein A1 (RPA1), DEAD-Box Helicase 6 (DDX6), Proteasome 20S Subunit Beta 2 (PSMB2), DnaJ Heat Shock Protein Family (HSP40) Member A1 (DNAJA1), Cyclin Dependent Kinase 2 (CDK2), and Tubulin Alpha 8 (TUBA8). Overexpression of  < 0.05) . DDX6 pl (Lpin1) . PSMB2 ce tissue . HSP40 pes (T2D) . Decreasin swine . m6A-dephibition . In Marthibition , the COPZNF543 were minimal between the high- and low-IMF groups, we inferred that although the editing sites in the 3′ UTR region of ZNF543 have miRNA–mRNA interaction gain/loss or change, the role of this interaction may have no effect on ZNF543 expression. There are possible post-translational effects that might justify an effect of ZNF543 on IMF. One transcript, named GHSR-202, showed a 10.59-fold change expression difference between the high- and low-IMF groups. We searched all of the potential interactions with miRNAs in PubMed. ssc-miR-15b, ssc-miR-16, ssc-miR-103, and ssc-miR-221-3p were reported to have different expressions in the myofiber between lean and obese pig breeds [GHSR are involved in obesity [GHSR being the center of the GO cluster, we speculated that GHSR may play a crucial part in IMF regulation. The editing site in the 3′ UTR of GHSR may contribute to the lipogenesis by regulating the miRNA of ssc-miR-15b, ssc-miR-16, ssc-miR-103, or ssc-miR-221-3p. To determine the target miRNA, further research, such as dual luciferase reporter assay should be performed.As the differences in the expression levels of the transcripts in g breeds . ssc-miRg breeds . ssc-miRg breeds . Ghrelin obesity ,56. CombACAT1, NCOA3, COPA, and especially GHSR, should be deeply studied in future research. More research such as RNA pulldown, western blotting and dual luciferase reporter assay should be done to explore the relationship between these candidate editing sites and interacted genes and proteins. Our findings provide some new RNA editing sites and some new insights of RNA editing function which useful for pig IMF breeding or human insulin resistances research.In this study, a total of 295 RNA editing sites were identified using strand-specific RNA and whole-genome DNA sequencing data in pig longissimus dorsi muscle. The functional analysis of the genes with hyper- and hypo-editing sites revealed the potentially functional importance of RNA editing in porcine IMF regulation. Some candidate editing sites, such as in