Long noncoding RNA pncRNA-D reduces cyclin D1 gene expression and arrests cell cycle through RNA m6A modification.

pncRNA-D is an irradiation-induced 602-nt long noncoding RNA transcribed from the promoter region of the cyclin D1 (CCND1) gene. CCND1 expression is predicted to be inhibited through an interplay between pncRNA-D and RNA-binding protein TLS/FUS. Because the pncRNA-D-TLS interaction is essential for pncRNA-D-stimulated CCND1 inhibition, here we studied the possible role of RNA modification in this interaction in HeLa cells. We found that osmotic stress induces pncRNA-D by recruiting RNA polymerase II to its promoter. pncRNA-D was highly m6A-methylated in control cells, but osmotic stress reduced the methylation and also arginine methylation of TLS in the nucleus. Knockdown of the m6A modification enzyme methyltransferase-like 3 (METTL3) prolonged the half-life of pncRNA-D, and among the known m6A recognition proteins, YTH domain-containing 1 (YTHDC1) was responsible for binding m6A of pncRNA-D Knockdown of METTL3 or YTHDC1 also enhanced the interaction of pncRNA-D with TLS, and results from RNA pulldown assays implicated YTHDC1 in the inhibitory effect on the TLS-pncRNA-D interaction. CRISPR/Cas9-mediated deletion of candidate m6A site decreased the m6A level in pncRNA-D and altered its interaction with the RNA-binding proteins. Of note, a reduction in the m6A modification arrested the cell cycle at the G0/G1 phase, and pncRNA-D knockdown partially reversed this arrest. Moreover, pncRNA-D induction in HeLa cells significantly suppressed cell growth. Collectively, these findings suggest that m6A modification of the long noncoding RNA pncRNA-D plays a role in the regulation of CCND1 gene expression and cell cycle progression.

m6A Modified Short RNA Fragments Inhibit Cytoplasmic TLS/FUS Aggregation Induced by Hyperosmotic Stress.

Translocated in LipoSarcoma/Fused in Sarcoma (TLS/FUS) is a nuclear RNA binding protein whose mutations cause amyotrophic lateral sclerosis. TLS/FUS undergoes LLPS and forms membraneless particles with other proteins and nucleic acids. Interaction with RNA alters conformation of TLS/FUS, which affects binding with proteins, but the effect of m6A RNA modification on the TLS/FUS-RNA interaction remains elusive. Here, we investigated the binding specificity of TLS/FUS to m6A RNA fragments by RNA pull down assay, and elucidated that both wild type and ALS-related TLS/FUS mutants strongly bound to m6A modified RNAs. TLS/FUS formed cytoplasmic foci by treating hyperosmotic stress, but the cells transfected with m6A-modified RNAs had a smaller number of foci. Moreover, m6A-modified RNA transfection resulted in the cells obtaining higher resistance to the stress. In summary, we propose TLS/FUS as a novel candidate of m6A recognition protein, and m6A-modified RNA fragments diffuse cytoplasmic TLS/FUS foci and thereby enhance cell viability.

Arginine methylation of translocated in liposarcoma (TLS) inhibits its binding to long noncoding RNA, abrogating TLS-mediated repression of CBP/p300 activity.

Translocated in liposarcoma (TLS) is an RNA-binding protein and a transcription-regulatory sensor of DNA damage. TLS binds promoter-associated noncoding RNA (pncRNA) and inhibits histone acetyltransferase (HAT) activity of CREB-binding protein (CBP)/E1A-binding protein P300 (p300) on the cyclin D1 (CCND1) gene. Although post-translational modifications of TLS, such as arginine methylation, are known to regulate TLS's nucleocytoplasmic shuttling and assembly in stress granules, its interactions with RNAs remain poorly characterized. Herein, using various biochemical assays, we confirmed the earlier observations that TLS is methylated by protein arginine methyltransferase 1 (PRMT1) in vitro The arginine methylation of TLS disrupted binding to pncRNA and also prevented binding of TLS to and inhibition of CBP/p300. This result indicated that arginine methylation of TLS abrogates both binding to pncRNA and TLS-mediated inhibition of CBP/p300 HAT activities. We also report that an arginine residue within the Arg-Gly-Gly domain of TLS, Arg-476, serves as the major determinant for binding to pncRNA. Either methylation or mutation of Arg-476 of TLS significantly decreased pncRNA binding and thereby prevented a pncRNA-induced allosteric alteration in TLS that is required for its interaction with CBP/p300. Moreover, unlike WT TLS, an R476A TLS mutant did not inhibit CCND1 promoter activity in luciferase reporter assays. Taken together, we propose the hypothesis that arginine methylation of TLS regulates both TLS-nucleic acid and TLS-protein interactions and thereby participates in transcriptional regulation.

Altered gene expression profiles of histone lysine methyltransferases and demethylases in rheumatoid arthritis synovial fibroblasts.

OBJECTIVES: Aberrant histone lysine methylation (HKM) has been reported in rheumatoid arthritis (RA) synovial fibroblasts (SFs). As histone lysine methyltransferases (HKMTs) and demethylases (HKDMs) regulate HKM, these enzymes are believed to be dysregulated in RASFs. The aim of this study is to clarify whether gene expressions of HKMTs and HKDMs are altered in RASFs. METHODS: SFs were isolated from synovial tissues obtained from RA or osteoarthritis (OA) patients during total knee joint replacement. The mRNA levels of 34 HKMTs and 22 HKDMs were examined after stimulation with tumour necrosis factor α (TNF-α) in RASFs and OASFs. RESULTS: The gene expression of the 12 HKMTs, including MLL1, MLL3, SUV39H1, SUV39H2, PRDM2, EZH2, SETD2, NSD2, NSD3, SMYD4, DOT1, and PR-set7, that catalyse the methylation of H3K4, H3K9, H3K27, H3K36, H3K79, or H4K20 was higher after TNFα stimulation in RASFs vs. OASFs. The gene expression of the 4 HKDMs, including FBXL10, NO66, JMJD2D, and FBXL11, that catalyse the methylation of H3K4, H3K9, or H3K36 was higher after TNFα stimulation in RASFs vs. OASFs. CONCLUSIONS: The study findings suggest that the HKM-modifying enzymes are involved in the alteration of HKM, which results in changes in the gene expression of RASFs.

Aberrant histone acetylation contributes to elevated interleukin-6 production in rheumatoid arthritis synovial fibroblasts.

Accumulating evidence indicates that epigenetic aberrations have a role in the pathogenesis of rheumatoid arthritis (RA). However, reports on histone modifications are as yet quite limited in RA. Interleukin (IL)-6 is an inflammatory cytokine which is known to be involved in the pathogenesis of RA. Here we report the role of histone modifications in elevated IL-6 production in RA synovial fibroblasts (SFs). The level of histone H3 acetylation (H3ac) in the IL-6 promoter was significantly higher in RASFs than osteoarthritis (OA) SFs. This suggests that chromatin structure is in an open or loose state in the IL-6 promoter in RASFs. Furthermore, curcumin, a histone acetyltransferase (HAT) inhibitor, significantly reduced the level of H3ac in the IL-6 promoter, as well as IL-6 mRNA expression and IL-6 protein secretion by RASFs. Taken together, it is suggested that hyperacetylation of histone H3 in the IL-6 promoter induces the increase in IL-6 production by RASFs and thereby participates in the pathogenesis of RA.

Induced ncRNAs allosterically modify RNA-binding proteins in cis to inhibit transcription.

With the recent recognition of non-coding RNAs (ncRNAs) flanking many genes, a central issue is to obtain a full understanding of their potential roles in regulated gene transcription programmes, possibly through different mechanisms. Here we show that an RNA-binding protein, TLS (for translocated in liposarcoma), serves as a key transcriptional regulatory sensor of DNA damage signals that, on the basis of its allosteric modulation by RNA, specifically binds to and inhibits CREB-binding protein (CBP) and p300 histone acetyltransferase activities on a repressed gene target, cyclin D1 (CCND1) in human cell lines. Recruitment of TLS to the CCND1 promoter to cause gene-specific repression is directed by single-stranded, low-copy-number ncRNA transcripts tethered to the 5' regulatory regions of CCND1 that are induced in response to DNA damage signals. Our data suggest that signal-induced ncRNAs localized to regulatory regions of transcription units can act cooperatively as selective ligands, recruiting and modulating the activities of distinct classes of RNA-binding co-regulators in response to specific signals, providing an unexpected ncRNA/RNA-binding protein-based strategy to integrate transcriptional programmes.

Id4, a new candidate gene for senile osteoporosis, acts as a molecular switch promoting osteoblast differentiation.

Excessive accumulation of bone marrow adipocytes observed in senile osteoporosis or age-related osteopenia is caused by the unbalanced differentiation of MSCs into bone marrow adipocytes or osteoblasts. Several transcription factors are known to regulate the balance between adipocyte and osteoblast differentiation. However, the molecular mechanisms that regulate the balance between adipocyte and osteoblast differentiation in the bone marrow have yet to be elucidated. To identify candidate genes associated with senile osteoporosis, we performed genome-wide expression analyses of differentiating osteoblasts and adipocytes. Among transcription factors that were enriched in the early phase of differentiation, Id4 was identified as a key molecule affecting the differentiation of both cell types. Experiments using bone marrow-derived stromal cell line ST2 and Id4-deficient mice showed that lack of Id4 drastically reduces osteoblast differentiation and drives differentiation toward adipocytes. On the other hand knockdown of Id4 in adipogenic-induced ST2 cells increased the expression of Ppargamma2, a master regulator of adipocyte differentiation. Similar results were observed in bone marrow cells of femur and tibia of Id4-deficient mice. However the effect of Id4 on Ppargamma2 and adipocyte differentiation is unlikely to be of direct nature. The mechanism of Id4 promoting osteoblast differentiation is associated with the Id4-mediated release of Hes1 from Hes1-Hey2 complexes. Hes1 increases the stability and transcriptional activity of Runx2, a key molecule of osteoblast differentiation, which results in an enhanced osteoblast-specific gene expression. The new role of Id4 in promoting osteoblast differentiation renders it a target for preventing the onset of senile osteoporosis.

Loop lengths of G-quadruplex structures affect the G-quadruplex DNA binding selectivity of the RGG motif in Ewing's sarcoma.

The G-quadruplex nucleic acid structural motif is a target for designing molecules with potential anticancer properties. To achieve therapeutic selectivity by targeting the G-quadruplex, the molecules must be able to differentiate between the DNA of different G-quadruplexes. We recently reported that the Arg-Gly-Gly repeat (RGG) of the C-terminus in Ewing's sarcoma protein (EWS), which is a group of dominant oncogenes that arise due to chromosomal translocations, is capable of binding to G-quadruplex telomere DNA and RNA via arginine residues and stabilize the G-quadruplex DNA form in vitro. Here, we show that the RGG of EWS binds preferentially to G-quadruplexes with longer loops, which is not related to the topology of the G-quadruplex structure. Moreover, the G-quadruplex DNA binding of the RGG in EWS depends on the phosphate backbone of the loops in the G-quadruplex DNA. We also investigated the G-quadruplex DNA binding activity of the N- and C-terminally truncated RGG to assess the role of the regions in the RGG in G-quadruplex DNA binding. Our findings indicate that the RGG and the other arginine-rich motif of residues 617-656 of the RGG in EWS are important for the specific binding to G-quadruplex DNA. These findings will contribute to the development of molecules that selectively target different G-quadruplex DNA.

Long noncoding RNA as a regulator for transcription.

Investigation of noncoding RNAs is in rapid progress, especially regarding translational repression by small (short) noncoding RNAs like microRNAs with 20-25 nucleotide-lengths, while long noncoding RNAs with nucleotide length of more than two hundred are also emerging. Indeed, our analysis has revealed that a long noncoding RNA transcribed from cyclin D1 promoter of 200 and 300 nucleotides exerts transcriptional repression through its binding protein TLS instead of translational repression. Translational repression is executed by short noncoding RNAs, while transcriptional repression is mainly done by long noncoding RNAs. These long noncoding RNAs are heterogeneous molecules and employ divergent molecular mechanisms to exert transcriptional repression. In this review, I overview recent publications regarding the transcription regulation by long noncoding RNAs and explore their biological significance. In addition, the relation between a random transcriptional activity of RNA polymerase II and the origin of long noncoding RNAs is discussed.

TLS and PRMT1 synergistically coactivate transcription at the survivin promoter through TLS arginine methylation.

TLS (Translocated in LipoSarcoma), also termed FUS, is a multifunctional protein implicated in diverse cellular events such as maintaining genome integrity and regulating gene expression. We have focused on the role of TLS as a coregulator in transcriptional regulation. In the process of investigating TLS-binding proteins, we found that PRMT1 (protein arginine methyltransferase 1) was in complex with TLS. We analyzed the methylation status of endogenous TLS and demonstrated that TLS was arginine-methylated by PRMT1. Using mass spectrometry, we identified that four arginine residues within TLS (R216, R218, R242 and R394) were consistently dimethylated. We performed luciferase reporter assays to assess the functional consequence of TLS arginine methylation in transcriptional regulation and, interestingly, observed that TLS and PRMT1 synergistically coactivated transcription at the survivin promoter. Further analysis using a catalytic-dead PRMT1 or methylation inhibitor both showed that the synergistic transcriptional activation was mediated by TLS arginine-methylation. These results revealed a cooperative role of TLS and PRMT1 in transcriptional regulation.

Promoter-associated long noncoding RNAs repress transcription through a RNA binding protein TLS.

The majority of the human genome is found to be transcribed and generates mostly noncoding (nc) RNAs that do not possess protein information. MicroRNAs are one of the well-identified small ncRNAs, but occupy merely a fraction of ncRNAs. Long (large) ncRNAs are emerging as a novel class of ncRNAs, but knowledge of these ncRNAs is far less accumulated. Long ncRNAs are tentatively classified as an ncRNA species containing more than 200 nucleotides. Recently, a long promoter-associated ncRNA (pncRNA) has been identified to be transcribed from the cyclin D1 promoter upon induction by genotoxic factors like ionizing-irradiation. The cyclin D1 pncRNA is specifically bound with an RNA-binding protein TLS (Translocated in liposarcoma) and exerts transcriptional repression through histone acetyltransferase (HAT) inhibitory activity. Analysis of TLS and the pncRNAs could provide a model for elucidating their roles inregulation of mammalian transcriptional programs. The pncRNA binding to TLS turns out to be an essential event for the HAT inhibitory activity. A key consensus sequence of the pncRNA is composed of GGUG, while not every RNA sequence bearing GGUG is targeted by TLS, suggesting that a secondary structure of the GGUG-bearing RNAs is also involved in recognition by TLS. Taken together, TLS is a unique mediator between signals of the long ncRNAs and transcription, suggesting that RNA networking functions in living cells.(1-3).

Non-coding RNA suppresses FUS aggregation caused by mechanistic shear stress on pipetting in a sequence-dependent manner.

Fused in sarcoma/translocated in liposarcoma (FUS/TLS) is a multitasking RNA/DNA binding protein. FUS aggregation is implicated in various neurodegenerative diseases. RNA was suggested to modulate phase transition of FUS. Here, we found that FUS transforms into the amorphous aggregation state as an instant response to the shear stress caused by usual pipetting even at a low FUS concentration, 100 nM. It was revealed that non-coding RNA can suppress the transformation of FUS into aggregates. The suppressive effect of RNA on FUS aggregation is sequence-dependent. These results suggested that the non-coding RNA could be a prospective suppressor of FUS aggregation caused by mechanistic stress in cells. Our finding might pave the way for more research on the role of RNAs as aggregation inhibitors, which could facilitate the development of therapies for neurodegenerative diseases.

Direct visualization of the conformational change of FUS/TLS upon binding to promoter-associated non-coding RNA.

High-speed AFM revealed the conformational change of fused in sarcoma (FUS) from a compact to an extended structure upon binding of non-coding RNA, which is supposed to allow FUS to bind to CBP/p300 for transcriptional interference. Thus, a mechanistic insight into transcription regulation by FUS and non-coding RNA is provided.

RNA sequence and length contribute to RNA-induced conformational change of TLS/FUS.

Translocated in liposarcoma (TLS)/fused in sarcoma (FUS) is a multitasking DNA/RNA binding protein implicated in cancer and neurodegenerative diseases. Upon DNA damage, TLS is recruited to the upstream region of the cyclin D1 gene (CCND1) through binding to the promotor associated non-coding RNA (pncRNA) that is transcribed from and tethered at the upstream region. Binding to pncRNA is hypothesized to cause the conformational change of TLS that enables its inhibitive interaction with histone acetyltransferases and resultant repression of CCND1 expression, although no experimental proof has been obtained. Here, the closed-to-open conformational change of TLS on binding pncRNA was implied by fluorescence resonance energy transfer. A small fragment (31 nucleotides) of the full-length pncRNA (602 nucleotides) was shown to be sufficient for the conformational change of TLS. Dissection of pncRNA identified the G-rich RNA sequence that is critical for the conformational change. The length of RNA was also revealed to be critical for the conformational change. Furthermore, it was demonstrated that the conformational change of TLS is caused by another target DNA and RNA, telomeric DNA and telomeric repeat-containing RNA. The conformational change of TLS on binding target RNA/DNA is suggested to be essential for biological functions.

Transcriptional regulation through noncoding RNAs and epigenetic modifications.

The recognition that substantial portions of the "noncoding (nc)" regions of metazoan genomes are transcribed has generated intense interest in the potential biological roles of ncRNAs. Gene silencing has emerged as one of the major functions of short double stranded ncRNAs (22-25 nucleotides, nt) that are generated by specific processing machinery. Transcription is also regulated by other classes of ncRNAs, including long (more than 200 nt), single-stranded, polyadenylated RNA molecules. Here, we review recent studies suggesting that some ncRNAs appear to function by binding to and regulating the activities of transcriptional co-activator or co-repressor complexes. Although the extents to which these mechanisms are used remains to be established, the identification of highly conserved ncRNAs and the presence of RNA binding domains in a large number of transcriptional co-regulators raise the possibility that ncRNA/coregulator interactions play broad roles in the regulation of gene expression.

Plastic roles of phenylalanine and tyrosine residues of TLS/FUS in complex formation with the G-quadruplexes of telomeric DNA and TERRA.

The length of a telomere is regulated via elongation and shortening processes. Telomeric DNA and telomeric repeat-containing RNA (TERRA), which both contain G-rich repeated sequences, form G-quadruplex structures. Previously, translocated in liposarcoma (TLS) protein, also known as fused in sarcoma (FUS) protein, was found to form a ternary complex with the G-quadruplex structures of telomeric DNA and TERRA. We then showed that the third RGG motif of TLS, the RGG3 domain, is responsible for the complex formation. However, the structural basis for their binding remains obscure. Here, NMR-based binding assaying revealed the interactions in the binary and ternary complexes of RGG3 with telomeric DNA or/and TERRA. In the ternary complex, tyrosine bound exclusively to TERRA, while phenylalanine bound exclusively to telomeric DNA. Thus, tyrosine and phenylalanine each play a central role in the recognition of TERRA and telomeric DNA, respectively. Surprisingly in the binary complexes, RGG3 used both tyrosine and phenylalanine residues to bind to either TERRA or telomeric DNA. We propose that the plastic roles of tyrosine and phenylalanine are important for RGG3 to efficiently form the ternary complex, and thereby regulate the telomere shortening.

Novel homeodomain-interacting protein kinase family member, HIPK4, phosphorylates human p53 at serine 9.

We describe here the cloning and characterization of a novel mouse homeodomain-interacting protein kinase (HIPK)-like gene, Hipk4. Hipk4 is expressed in lung and in white adipose tissue and encodes a 616 amino acid protein that includes a serine/threonine kinase domain. We demonstrate that HIPK4 could phosphorylate human p53 protein at serine 9, both in vitro and in vivo. Among known p53-responsive promoters, activity of the human survivin promoter, which is repressed by p53, was decreased by HIPK4 in p53 functional A549 cells. Human BCL2-associated X protein-promoter activity was not affected. These findings suggest that phosphorylation of p53 at serine 9 is important for p53 mediated transcriptional repression.

The binding specificity of Translocated in LipoSarcoma/FUsed in Sarcoma with lncRNA transcribed from the promoter region of cyclin D1.

BACKGROUND: Translocated in LipoSarcoma (TLS, also known as FUsed in Sarcoma) is an RNA/DNA binding protein whose mutation cause amyotrophic lateral sclerosis. In previous study, we demonstrated that TLS binds to long noncoding RNA, promoter-associated ncRNA-D (pncRNA-D), transcribed from the 5' upstream region of cyclin D1 (CCND1), and inhibits the expression of CCND1. RESULTS: In order to elucidate the binding specificity between TLS and pncRNA-D, we divided pncRNA-D into seven fragments and examined the binding with full-length TLS, TLS-RGG2-zinc finger-RGG3, and TLS-RGG3 by RNA pull down assay. As a result, TLS was able to bind to all the seven fragments, but the fragments containing reported recognition motifs (GGUG and GGU) tend to bind more solidly. The full-length TLS and TLS-RGG2-zinc finger-RGG3 showed a similar interaction with pncRNA-D, but the binding specificity of TLS-RGG3 was lower compared to the full-length TLS and TLS-RGG2-zinc finger-RGG3. Mutation in GGUG and GGU motifs dramatically decreased the binding, and unexpectedly, we could only detect weak interaction with the RNA sequence with stem loop structure. CONCLUSION: The binding of TLS and pncRNA-D was affected by the presence of GGUG and GGU sequences, and the C terminal domains of TLS function in the interaction with pncRNA-D.

Histone Methylation and STAT-3 Differentially Regulate Interleukin-6-Induced Matrix Metalloproteinase Gene Activation in Rheumatoid Arthritis Synovial Fibroblasts.

OBJECTIVE: Synovial fibroblasts (SFs) produce matrix-degrading enzymes that cause joint destruction in rheumatoid arthritis (RA). Epigenetic mechanisms play a pivotal role in autoimmune diseases. This study was undertaken to elucidate the epigenetic mechanism that regulates the transcription of matrix metalloproteinases (MMPs) in RASFs. METHODS: MMP gene expression and histone methylation profiles in the MMP promoters were examined in RASFs. The effect of WD repeat domain 5 (WDR5) silencing on histone methylation and MMP gene expression in RASFs was analyzed. MMP gene expression, surface expression of the interleukin-6 (IL-6) receptor, phosphorylation of STAT-3, and binding of STAT-3 in the MMP promoters were investigated in RASFs stimulated with IL-6. RESULTS: The MMP-1, MMP-3, MMP-9, and MMP-13 genes were actively transcribed in RASFs. Correspondingly, the level of histone H3 trimethylated at lysine 4 (H3K4me3) was elevated, whereas that of H3K27me3 was suppressed in the MMP promoters in RASFs. The decrease in H3K4me3 via WDR5 small interfering RNA reduced the levels of messenger RNA for MMP-1, MMP-3, MMP-9, and MMP-13 in RASFs. Interestingly, IL-6 signaling significantly increased the expression of MMP-1, MMP-3, and MMP-13, but not MMP-9, in RASFs. Although the IL-6 signaling pathway was similarly active in RASFs and osteoarthritis SFs, STAT-3 bound to the MMP-1, MMP-3, and MMP-13 promoters, but not the MMP-9 promoter, after IL-6 stimulation in RASFs. CONCLUSION: Our findings indicate that histone methylation and STAT-3 regulate spontaneous and IL-6-induced MMP gene activation in RASFs. The combination of chromatin structure and transcription factors may regulate distinct arthritogenic properties of RASFs.

Development of a mouse monoclonal antibody for the detection of asymmetric dimethylarginine of Translocated in LipoSarcoma/FUsed in Sarcoma and its application in analyzing methylated TLS.

BACKGROUND: RNA-binding protein Translocated in LipoSarcoma/FUsed Sarcoma (TLS/FUS) is one of causative genes for familial amyotrophic lateral sclerosis (ALS). We previously identified that TLS was associated with protein arginine methyltransferase 1 (PRMT1), and four arginine residues within TLS (R216, R218, R242 and R394) were consistently dimethylated. Protein arginine methylation is involved in various cellular events such as signal transduction, transcriptional regulation and protein-protein interactions. RESULTS: To understand the biological role of arginine methylation of RNA-binding protein, we prepared and characterized a mouse monoclonal antibody against asymmetric dimethylarginine of TLS. By cloning and screening, one stable hybridoma cell clone (2B12) producing anti-asymmetric dimethylated TLS on R216 and R218 antibody was established. The monoclonal antibody 2B12 is specific for the asymmetrically dimethylated arginine peptide and does not react with the same peptide sequence containing unmodified and symmetrically dimethylated arginine residues by dot-blot analysis. 2B12 was also validated GST tagged TLS with PRMT1 by in vitro arginine methylation assays. Since methylated TLS in HeLa cells and mouse and human brain protein extracts was immunoprecipitated with 2B12, we performed RNA-binding protein immunoprecipitation assays using HeLa cell lysate and this antibody. We demonstrated that the long noncoding RNA (lncRNA) transcribed from cyclin D1 promoter binds methylated TLS. CONCLUSIONS: A monoclonal antibody that is capable of detecting the methylarginine status of TLS will facilitate the molecular and cellular analysis of transcriptional regulation by lncRNA through methylated TLS, and can be used as a favorable tool for clinical diagnosis of ALS caused by TLS dysregulation.