microRNA Biogenesis Research Articles

Visualizing a protonated RNA state that modulates microRNA-21 maturation.

MicroRNAs are evolutionarily conserved small, noncoding RNAs that regulate diverse biological processes. Due to their essential regulatory roles, microRNA biogenesis is tightly regulated, where protein factors are often found to interact with specific primary and precursor microRNAs for regulation. Here, using NMR relaxation dispersion spectroscopy and mutagenesis, we reveal that the precursor of oncogenic microRNA-21 exists as a pH-dependent ensemble that spontaneously reshuffles the secondary structure of the entire apical stem-loop region, including the Dicer cleavage site. We show that the alternative excited conformation transiently sequesters the bulged adenine into a noncanonical protonated A+-G mismatch, conferring a substantial enhancement in Dicer processing over its ground conformational state. These results indicate that microRNA maturation efficiency may be encoded in the intrinsic dynamic ensemble of primary and precursor microRNAs, providing a potential means of regulating microRNA biogenesis in response to environmental and cellular stimuli.

Identification of key sequence features required for microRNA biogenesis in plants

MicroRNAs (miRNAs) are endogenous small RNAs of ∼21 nt that regulate multiple biological pathways in multicellular organisms. They derive from longer transcripts that harbor an imperfect stem-loop structure. In plants, the ribonuclease type III DICER-LIKE1 assisted by accessory proteins cleaves the precursor to release the mature miRNA. Numerous studies highlight the role of the precursor secondary structure during plant miRNA biogenesis; however, little is known about the relevance of the precursor sequence. Here, we analyzed the sequence composition of plant miRNA primary transcripts and found specifically located sequence biases. We show that changes in the identity of specific nucleotides can increase or abolish miRNA biogenesis. Most conspicuously, our analysis revealed that the identity of the nucleotides at unpaired positions of the precursor plays a crucial role during miRNA biogenesis in Arabidopsis.

Establishment and characterization of VOA1066 cells: An undifferentiated endometrial carcinoma cell line.

Dedifferentiated endometrial carcinoma (DDEC) is a rare but highly aggressive type of endometrial cancer, in which an undifferentiated carcinoma arises from a low-grade endometrioid endometrial carcinoma. The low-grade component is often eclipsed, likely due to an outgrowth of the undifferentiated component, and the tumor may appear as a pure undifferentiated endometrial carcinoma (UEC). We and others have recently identified inactivating mutations of SMARCA4, SMARCB1 or ARID1B, subunits of the SWI/SNF chromatin-remodeling complex, that are unique to the undifferentiated component and are present in a large portion of DDEC and UEC. However, the understanding of whether and how these mutations drive cancer progression and histologic dedifferentiation is hindered by lack of cell line models of DDEC or UEC. Here, we established the first UEC cell line, VOA1066, which is highly tumorigenic in vivo. This cell line has a stable genome with very few somatic mutations, which do include inactivating mutations of ARID1A and ARID1B (2 mutations each), and a heterozygous hotspot DICER1 mutation in its RNase IIIb domain. Immunohistochemistry staining confirmed the loss of ARID1B, but ARID1A staining was retained due to the presence of a truncating non-functional ARID1A protein. The heterozygous DICER1 hotspot mutation has little effect on microRNA biogenesis. No additional DICER1 hotspot mutations have been identified in a cohort of 33 primary tumors. Therefore, we have established the first UEC cell line with dual inactivation of both ARID1A and ARID1B as the main genomic feature. This cell line will be useful for studying the roles of ARID1A and ARID1B mutations in the development of UEC.

MicroRNA-Independent Modulation of DICER1 Expression by hAgo2.

Many proteins, including DICER1 and hAgo2, are involved in the biogenesis of microRNAs (miRNAs). Whether hAgo2 regulates DICER1 expression is unknown. Exogenously overexpressed hAgo2 suppressed DICER1 expression at the levels of both protein and mRNA, and the reduction in hAgo2 expression enhanced DICER1 expression. Precursor miRNA processing mediated by DICER1 was also modulated by hAgo2. However, hAgo2 protein did not suppress DICER1 promoter activity. Therefore, hAgo2 protein probably regulates DICER1 expression at the posttranscriptional level. Indeed, hAgo2 protein inhibited the reporter assay of the DICER1 mRNA 3' untranslated region (3'-UTR). Previous reports have demonstrated that miRNAs (e.g., let-7 and miR-103/107) inhibited DICER1 expression posttranscriptionally. However, hAgo2 still suppressed DICER1 expression in the cells depleted of these miRNAs. Moreover, the reporter activities of the DICER1 mRNA 3'-UTR without these miRNA binding sites were still suppressed by hAgo2. Therefore, in addition to an miRNA-dependent pathway, hAgo2 can also modulate DICER1 expression through an miRNA-independent mechanism. Downregulation of DICER1 expression was further proven to be dependent on both hAgo2 and AUF1 proteins. Interactions of hAgo2 and AUF1 proteins were demonstrated by the coimmunoprecipitation assay. As expected, hAgo2 could not suppress the DICER1 mRNA 3'-UTR reporter with a mutation in the potential AUF1-binding site. Thus, downregulation of DICER1 expression through the 3'-UTR requires both hAgo2 and AUF1.

MicroRNA biogenesis in plant

MicroRNAs (miRNAs), a type of endogenous non-coding small RNA, with a length of 20 to 24 nt, represses their target gene expression post-transcriptionally. There are a growing number of studies have discovered that miRNAs are found in animals and plants. Plant miRNAs are involved in growth and development, and they play an important role in physiological and biochemical regulation. This review mainly focuses on components of different miRNA processing pathways and how miRNA effectors inhibit the expression of target genes in plants. We discuss not only the various pathways affecting miRNA biogenesis, but also the different forms of miRNA effector pathways. Previous studies found that there have been some primary advances in the formation of miRNAs, we hope to provide a comprehensive introduction to the biogenesis of plant miRNAs.

Dynamic changes in DICER levels in adipose tissue control metabolic adaptations to exercise

DICER is a key enzyme in microRNA (miRNA) biogenesis. Here we show that aerobic exercise training up-regulates DICER in adipose tissue of mice and humans. This can be mimicked by infusion of serum from exercised mice into sedentary mice and depends on AMPK-mediated signaling in both muscle and adipocytes. Adipocyte DICER is required for whole-body metabolic adaptations to aerobic exercise training, in part, by allowing controlled substrate utilization in adipose tissue, which, in turn, supports skeletal muscle function. Exercise training increases overall miRNA expression in adipose tissue, and up-regulation of miR-203-3p limits glycolysis in adipose under conditions of metabolic stress. We propose that exercise training-induced DICER-miR-203-3p up-regulation in adipocytes is a key adaptive response that coordinates signals from working muscle to promote whole-body metabolic adaptations.

MAC5, an RNA-binding protein, protects pri-miRNAs from SERRATE-dependent exoribonuclease activities

MAC5 is a component of the conserved MOS4-associated complex. It plays critical roles in development and immunity. Here we report that MAC5 is required for microRNA (miRNA) biogenesis. MAC5 interacts with Serrate (SE), which is a core component of the microprocessor that processes primary miRNA transcripts (pri-miRNAs) into miRNAs and binds the stem-loop region of pri-miRNAs. MAC5 is essential for both the efficient processing and the stability of pri-miRNAs. Interestingly, the reduction of pri-miRNA levels in mac5 is partially caused by XRN2/XRN3, the nuclear-localized 5'-to-3' exoribonucleases, and depends on SE. These results reveal that MAC5 plays a dual role in promoting pri-miRNA processing and stability through its interaction with SE and/or pri-miRNAs. This study also uncovers that pri-miRNAs need to be protected from nuclear RNA decay machinery, which is connected to the microprocessor.

MicroRNA-135a-5p Promotes the Functional Recovery of Spinal Cord Injury by Targeting SP1 and ROCK

Emerging evidence indicates that microRNAs play a pivotal role in neural remodeling after spinal cord injury (SCI). This study aimed to investigate the mechanisms of miR-135a-5p in regulating the functional recovery of SCI by impacting its target genes and downstream signaling. The gene transfection assay and luciferase reporter assay confirmed the target relationship between miR-135a-5p and its target genes (specificity protein 1 [SP1] and Rho-associated kinase [ROCK]1/2). By establishing the H2O2-induced injury model, miR-135a-5p transfection was found to inhibit the apoptosis of PC12 cells by downregulating the SP1 gene, which subsequently induced downregulation of pro-apoptotic proteins (Bax, cleaved caspase-3) and upregulation of anti-apoptotic protein Bcl-2. By measuring the neurite lengths of PC12 cells, miR-135a-5p transfection was found to promote axon outgrowth by downregulating the ROCK1/2 gene, which subsequently caused upregulation of phosphate protein kinase B (AKT) and phosphate glycogen synthase kinase 3β (GSK3β). Use of the rat SCI models showed that miR-135a-5p could increase the Basso, Beattie, and Bresnahan (BBB) scores, indicating neurological function recovery. In conclusion, the miR-135a-5p-SP1-Bax/Bcl-2/caspase-3 and miR-135a-5p-ROCK-AKT/GSK3β axes are involved in functional recovery of SCI by regulating neural apoptosis and axon regeneration, respectively, and thus can be promising effective therapeutic strategies in SCI.

Correction for Kincaid et al., "Noncanonical MicroRNA (miRNA) Biogenesis Gives Rise to Retroviral Mimics of Lymphoproliferative and Immunosuppressive Host miRNAs".

Volume 5, no. 2, e00074-14, 2014, https://doi.org/10.1128/mBio.00074-14. R. P. Kincaid, Y. …

Abstract 549: Genome-wide CRISPR-Cas9 screen and RNAseq analysis identify new candidate synthetic lethality partners to PARP inhibitor in triple-negative breast cancer

Abstract As monotherapy, Poly (ADP-ribose) polymerase inhibitors (PARPi) have achieved remarkable success in treating tumors with germline BRCA1/2 mutation due to the synthetic lethality in DNA damage response. The efficacy of PARPi in combination with other therapeutic agents is still under investigation. However, BRCA mutant tumors constitute only 5-10% of total breast cancer diagnoses. Therefore, to extend the benefit of PARPi beyond BRCA mutant tumors, it is imperative to identify other genetic determinants that also contribute to PARPi sensitivity. Such knowledge also provides valuable insight into the development of combination therapies involving PARPi. To identify genes and pathways that are essential for cell survival under PARPi treatment, we performed genome-wide CRISPR-Cas9 knockout screens in a BRCA-functional, triple-negative breast cancer cell line MDA-MB-231 treated with talazoparib at a low dose (IC20, 20nM). Toronto CRISPR human knockout library TKOV3 was introduced into cells using lentivirus at MOI of 0.3~0.4. After puromycin selection, the surviving cells were treated with talazoparib or DMSO for 20 days before harvesting. The sgRNAs were sequenced at ~30 million reads per sample to achieve a 300x coverage over the TKOV3 library. To increase the accuracy of the CRISPR-Cas9 screen result, and to capture the transcriptomic changes during a relative long-term PARPi treatment as used in the screening process, we also did RNAseq profiling in MDA-MD-231 cells cultured in the conditions matched with the CRISPR-Cas9 screen. Using mRNA level as a filter to remove non-expressed genes, we were able to generate a list of candidate genes that have the potential to form synthetic lethal partnership with talazoparib. Our negative selection screen confirmed that loss of key components of DNA damage repair and DNA replication pathways such as ATM, RNASEH2C, ESCO2, EME1, and several components of Fanconi Anemia (FA) core complex sensitizes cells to talazoparib, as has been reported in similar screens using other PARP inhibitors. Meanwhile, our analysis also revealed several previously unrecognized partner genes involved in subcellular trafficking, RNA splicing, and microRNA biogenesis. In addition, the RNAseq profile depicted a transcriptomic response to long-term talazoparib treatment that has many distinctions from the essential pathways shown in the CRISPR-Cas9 screen, suggesting a complex, multi-layer regulation system in cell response to PARP inhibition. Our study identified a set of genes that have potential synthetic lethal interaction with PARPi. The status of these genes can be used to identify subset of triple-negative breast cancer patients who could potentially benefit from PARPi treatment. Strategies targeting these genes represent new opportunities for PARPi combination therapies. Citation Format: Xue Wu, Yue Zhao, Xiaoyu Xie, Xiaoling Xuei, Yunlong Liu, Lijun Cheng, Lang Li. Genome-wide CRISPR-Cas9 screen and RNAseq analysis identify new candidate synthetic lethality partners to PARP inhibitor in triple-negative breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 549.

Analyzing the Role of DICER1 Germline Variations in Papillary Thyroid Carcinoma

Introduction: DICER1 is a member of RNase III family that has a pivotal role in the biogenesis of microRNAs, being important for normal development. Dysregulation of DICER1 has been described in different human tumours; however, there is insufficient data on the risk of thyroid cancer in the presence of germline DICER1 variants, particularly when focusing on the background of papillary thyroid carcinoma (PTC). For this purpose, we ascertained the presence of DICER1 variants in 502 (PTC) cases available from The Cancer Genome Atlas (TCGA) research network in a well-characterized pathological context. Material and Methods: in this study we analyzed 502 samples from 502 patients, described as PTC in the TCGA database. Tumour diagnoses were re-evaluated by 2 pathologists (S.C. and M.S.-S.) on slides available from the database, and clinicopathological and demographic data was examined. Data concerning germline and sporadic DICER1 gene variants as well as frequent mutations in the genes involved in thyroid carcinogenesis (e.g., RAS and BRAFV600E) was retrieved from the database. Results and Discussion: We report 1 new germline possibly pathogenic variant, besides 15 others already been identified in ClinVar. We found that the DICER1-positive PTC group more frequently includes PTC variants, namely the oncocytic, follicular, and aggressive (hobnail variant of PTC) variants. A previous association of DICER1 had been demonstrated, mainly with the follicular variant of PTC and follicular thyroid carcinomas. Tumours harbouring germline DICER1 mutations were more frequently “bilateral” and “encapsulated.” The frequent association of DICER1 germline variants with other mutations associated with thyroid cancer can reflect an haploinsufficiency tumour suppressor gene function of DICER1, as suggested from the study of animal models.

MicroRNAs are indispensable for the proliferation and differentiation of adult neural progenitor cells in mice

More than two decades after the discovery of adult neurogenesis in humans, researchers still struggle to elucidate the underlying transcriptional and post-transcriptional mechanisms. RNA interference is a crucially important process in the central nervous system, and its role in adult neurogenesis is poorly understood. In this work, we address the role of Dicer-dependent microRNA biogenesis in neuronal differentiation of adult neural stem cells within the subventricular zone of the mouse brain. Loss of the Dicer1 gene in the tailless (Tlx)-positive cells did not cause the decline in their numbers, but severely affected differentiation. Thus, our findings identify yet another phenomenon associated with microRNA pathway deregulation in adult neural stem cells which might be of relevance both for neuroscience and clinical practice.

Knock-down of δ-aminolevulinic acid dehydratase via virus-induced gene silencing alters the microRNA biogenesis and causes stress-related reactions in citrus plants

The δ-aminolevulinic acid (δ-ALA) is an intermediate in the biosynthetic pathway of tetrapyrroles. Tetrapyrroles play vital roles in many biological processes such as photosynthesis, respiration, and light-sensing. ALA-dehydratase (ALAD) combines two molecules of δ-ALA to form porphobilinogen. In citrus, the silencing of ALAD caused discrete yellow spots and necrosis in leaves and stems. Additionally, it caused rapid death in developing new shoots. Herein, we hypothesize that the accumulation of δ-ALA results in severe stress and reduced meristem development. For that reason, we investigated the dynamic changes in the expression profiles of 23 microRNA (miRNA) identified through small RNA sequencing, from CTV‐tALAD plants in comparison with healthy C. macrophylla and C. macrophylla infiltrated with CTV-wt. Furthermore, we reported the effect of ALAD silencing on the total phenolics, H2O2, and reactive oxygen species (ROS) levels, to examine the possibilities of miRNAs involving the regulation of these pathways. Our results showed that the total phenolics content, H2O2, and O2− levels were increased in CTV-tALAD plants. Moreover, 63 conserved miRNA members belonging to 23 different miRNA families were differentially expressed in CTV-tALAD plants compared to controls. The identified miRNAs are implicated in auxin biosynthesis and signaling, axillary shoot meristem formation and leaf morphology, starch metabolism, and oxidative stress. Collectively, our findings suggested that ALAD silencing initiates stress on citrus plants. As a result, CTV-tALAD plants exhibit reduced metabolic rate, growth, and development in order to cope with the stress that resulted from the accumulation of δ-ALA. This cascade of events led to leaf, stem, and meristem necrosis and failure of new shoot development.

Linking key steps of microRNA biogenesis by TREX-2 and the nuclear pore complex in Arabidopsis.

SummaryUnlike in metazoans, the stepwise biogenesis of microRNAs (miRNAs) occurs within the nucleus in plants. Whether or how the major steps in miRNA biogenesis are coordinated is largely unknown. Here, we show that the plant TREX-2 complex promotes multiple steps in miRNA biogenesis, including transcription, processing and nuclear export. Core subunits of TREX-2, THP1 and SAC3A, interact and co-localize with RNA Polymerase II to promote the transcription of MIR genes in the nucleoplasm. TREX-2 interacts with the microprocessor component SERRATE and promotes the formation of Dicing bodies in the nucleoplasm. THP1 also interacts and co-localizes with the nucleoporin protein NUP1 at the nuclear envelope. NUP1 and THP1 promote the nuclear export of miRNAs and AGO1. These results suggest that TREX-2 coordinates the transcription, processing and export steps in miRNA biogenesis to ensure efficient miRNA production.

Determination of primary microRNA processing in clinical samples by targeted pri-miR-sequencing

MicroRNA expression is important for gene regulation and deregulated microRNA expression is often observed in diseases such as cancer. The processing of primary microRNA transcripts is an important regulatory step in microRNA biogenesis. Due to low expression level and association with chromatin, primary microRNAs are challenging to study in clinical samples where input material is limited. Here, we present a high-sensitivity targeted method to determine processing efficiency of several hundred primary microRNAs from total RNA that requires relatively few RNA sequencing reads. We validate the method using RNA from HeLa cells and show the applicability to clinical samples by analyzing RNA from normal liver and hepatocellular carcinoma. We identify 24 primary microRNAs with significant changes in processing efficiency from normal liver to hepatocellular carcinoma, among those the highly expressed miRNA-122 and miRNA-21, demonstrating that differential processing of primary microRNAs is occurring and could be involved in disease. With our method presented here we provide means to study pri-miRNA processing in disease from clinical samples.

Hyponastic Leaves 1 protects pri-miRNAs from nuclear exosome attack

Biogenesis of plant microRNAs (miRNAs) takes place in nuclear dicing bodies (D-bodies), where the ribonulease III-type enzyme Dicer-like 1 (DCL1) processes primary transcripts of miRNAs (pri-miRNAs) into miRNA/miRNA* (*, passenger strand) duplexes from either base-to-loop or loop-to-base directions. Hyponastic Leaves 1 (HYL1), a double-stranded RNA-binding protein, is crucial for efficient and accurate processing. However, whether HYL1 has additional function remains unknown. Here, we report that HYL1 plays a noncanonical role in protecting pri-miRNAs from nuclear exosome attack in addition to ensuring processing. Loss of functions in SOP1 or HEN2, two cofactors of the nucleoplasmic exosome, significantly suppressed the morphological phenotypes of hyl1-2 Remarkably, mature miRNAs generated from loop-to-base processing were partially but preferentially restored in the hyl1 sop1 and hyl1 hen2 double mutants. Accordingly, loop-to-base-processed pri-miRNAs accumulated to higher levels in double mutants. In addition, dysfunction of HEN2, but not of SOP1, in hyl1-2 resulted in overaccumulation of many base-to-loop-processed pri-miRNAs, with most of their respective miRNAs unaffected. In summary, our findings reveal an antagonistic action of exosome in pri-miRNA biogenesis and uncover dual roles of HYL1 in stabilizing and processing of pri-miRNAs.

Apple SERRATE negatively mediates drought resistance by regulating MdMYB88 and MdMYB124 and microRNA biogenesis

The function of serrate (SE) in miRNA biogenesis in Arabidopsis is well elucidated, whereas its role in plant drought resistance is largely unknown. In this study, we report that MdSE acts as a negative regulator of apple (Malus × domestica) drought resistance by regulating the expression levels of MdMYB88 and MdMYB124 and miRNAs, including mdm-miR156, mdm-miR166, mdm-miR172, mdm-miR319, and mdm-miR399. MdSE interacts with MdMYB88 and MdMYB124, two positive regulators of apple drought resistance. MdSE decreases the transcript and protein levels of MdMYB88 and MdMYB124, which directly regulate the expression of MdNCED3, a key enzyme in abscisic acid (ABA) biosynthesis. Furthermore, MdSE is enriched in the same region of the MdNECD3 promoter where MdMYB88/MdMYB124 binds. Consistently, MdSE RNAi transgenic plants are more sensitive to ABA-induced stomatal closure, whereas MdSE OE plants are less sensitive. In addition, under drought stress, MdSE is responsible for the biogenesis of mdm-miR399, a negative regulator of drought resistance, and negatively regulates miRNAs, including mdm-miR156, mdm-miR166, mdm-miR172, and mdm-miR319, which are positive regulators of drought resistance. Taken together, by revealing the negative role of MdSE, our results broaden our understanding of the apple drought response and provide a candidate gene for apple drought improvement through molecular breeding.

LIN28B regulates transcription and potentiates MYCN-induced neuroblastoma through binding to ZNF143 at target gene promotors.

LIN28B is highly expressed in neuroblastoma and promotes tumorigenesis, at least, in part, through inhibition of let-7 microRNA biogenesis. Here, we report that overexpression of either wild-type (WT) LIN28B or a LIN28B mutant that is unable to inhibit let-7 processing increases the penetrance of MYCN-induced neuroblastoma, potentiates the invasion and migration of transformed sympathetic neuroblasts, and drives distant metastases in vivo. Genome-wide chromatin immunoprecipitation coupled with massively parallel DNA sequencing (ChIP-seq) and coimmunoprecipitation experiments show that LIN28B binds active gene promoters in neuroblastoma cells through protein-protein interaction with the sequence-specific zinc-finger transcription factor ZNF143 and activates the expression of downstream targets, including transcription factors forming the adrenergic core regulatory circuitry that controls the malignant cell state in neuroblastoma as well as GSK3B and L1CAM that are involved in neuronal cell adhesion and migration. These findings reveal an unexpected let-7-independent function of LIN28B in transcriptional regulation during neuroblastoma pathogenesis.

Decoding Novel Mechanisms and Emerging Therapeutic Strategies in Breast Cancer Resistance.

Breast cancer (BC), an intricate and highly heterogeneous disorder, has presently afflicted 2.09 million females globally. Chemoresistance remains a paramount challenge in the treatment of BC. Owing to its assorted nature, the chemoresistant mechanisms of BC still need intensive research. Accumulating evidence suggests that abnormalities related to the biogenesis of cancer stem cells (CSCs) and microRNAs (miRNAs) are associated with BC progression and chemoresistance. The presently available interventions are inadequate to target chemoresistance, therefore more efficient alternatives are urgently needed to improvise existing therapeutic regimens. A myriad of strategies is being explored, such as immunotherapy, gene therapy, and combination treatment to surmount chemoresistance. Additionally, nanoparticles as chemotherapeutic carriers put forward the options to encapsulate numerous drugs, alone as well as in combination for cancer theranostics. This review summarizes the chemoresistance mechanisms of miRNAs and CSCs as well as the most recently documented therapeutic approaches for the treatment of chemoresistance in BC. By unraveling the underpinning mechanism of BC chemoresistance, researchers could possibly develop more efficient treatment strategies towards BC.

Interactome Analysis Illustrates Diverse Gene Regulatory Processes Associated with LIN28A in Human iPS Cell-Derived Neural Progenitor Cells

A single protein can be multifaceted, playing different roles depending on the cellular contexts and interacting molecules. LIN28A is a multi-functional RNA-binding protein that governs developmental timing, cell fate determination, stem cell pluripotency, cellular growth, and metabolism. LIN28A is best known as a post-transcriptional regulator of let-7 microRNA biogenesis, but its other roles have been increasingly recognized and studied. Although LIN28A-binding RNAs have been extensively analyzed, protein complexes associated with LIN28A have not been comprehensively studied. To more thoroughly understand the molecular roles of LIN28A, we profiled the endogenous LIN28A-interacting proteins using immunoprecipitation and liquid chromatography-tandem mass spectrometry. In order to enlighten its roles during neural development, we used neural progenitor cells differentiated from human induced pluripotent stem cells. We identified more than 400 LIN28A-interacting proteins, including 149 RNA-independent interactors. These interactors include proteins involved in RNA splicing and translation, as well as endoplasmic reticulum- or stress granule-localizing proteins, SWI/SNF chromatin remodeling complex. In addition, interaction with CARM1 methyltransferase led us to discover an arginine methylation of LIN28A. Overall, our analysis opens a new avenue for studying LIN28A’s gene regulatory roles including those in epigenetic regulation.