Classical and noncanonical functions of miRNAs in cancers.

Alterations in microRNAs (miRNAs) expression are causative in the initiation and progression of human cancers. The molecular events responsible for the widespread differential expression of miRNAs in malignancy are exemplified by their location in cancer-associated genomic regions, epigenetic mechanisms, transcriptional dysregulation, chemical modifications and editing, and alterations in miRNA biogenesis proteins. The classical miRNA function is synonymous with post-transcriptional repression of target protein genes. However, several studies have reported miRNAs functioning outside this paradigm and some of these novel modes of regulation of gene expression have been implicated in cancers. Here, we summarize key aspects of miRNA involvement in cancer, with a special focus on these lesser-studied mechanisms of action.

Intramolecular ligation method (iLIME) for pre-miRNA quantification and sequencing.

Hairpin-containing pre-miRNAs, produced from pri-miRNAs, are precursors of miRNAs (microRNAs) that play essential roles in gene expression and various human diseases. Current qPCR-based methods used to quantify pre-miRNAs are not effective to discriminate between pre-miRNAs and their parental pri-miRNAs. Here, we developed the intramolecular ligation method (iLIME) to quantify and sequence pre-miRNAs specifically. This method utilizes T4 RNA ligase 1 to convert pre-miRNAs into circularized RNAs, allowing us to design PCR primers to quantify pre-miRNAs, but not their parental pri-miRNAs. In addition, the iLIME also enables us to sequence the ends of pre-miRNAs using next-generation sequencing. Therefore, this method offers a simple and effective way to quantify and sequence pre-miRNAs, so it will be highly beneficial for investigating pre-miRNAs when addressing research questions and medical applications.

Genome-wide discovery of pre-miRNAs: comparison of recent approaches based on machine learning.

MOTIVATION: The genome-wide discovery of microRNAs (miRNAs) involves identifying sequences having the highest chance of being a novel miRNA precursor (pre-miRNA), within all the possible sequences in a complete genome. The known pre-miRNAs are usually just a few in comparison to the millions of candidates that have to be analyzed. This is of particular interest in non-model species and recently sequenced genomes, where the challenge is to find potential pre-miRNAs only from the sequenced genome. The task is unfeasible without the help of computational methods, such as deep learning. However, it is still very difficult to find an accurate predictor, with a low false positive rate in this genome-wide context. Although there are many available tools, these have not been tested in realistic conditions, with sequences from whole genomes and the high class imbalance inherent to such data. RESULTS: In this work, we review six recent methods for tackling this problem with machine learning. We compare the models in five genome-wide datasets: Arabidopsis thaliana, Caenorhabditis elegans, Anopheles gambiae, Drosophila melanogaster, Homo sapiens. The models have been designed for the pre-miRNAs prediction task, where there is a class of interest that is significantly underrepresented (the known pre-miRNAs) with respect to a very large number of unlabeled samples. It was found that for the smaller genomes and smaller imbalances, all methods perform in a similar way. However, for larger datasets such as the H. sapiens genome, it was found that deep learning approaches using raw information from the sequences reached the best scores, achieving low numbers of false positives. AVAILABILITY: The source code to reproduce these results is in: http://sourceforge.net/projects/sourcesinc/files/gwmirna Additionally, the datasets are freely available in: https://sourceforge.net/projects/sourcesinc/files/mirdata.

Structural Perspective into the Interaction of an Oncogenesis-Relevant pre-miRNA G-Quadruplex Ligand Carrier with the Protein Nucleolin.

The structural determinants of the interaction of the G-quadruplex (G4) motif found in precursor miRNA 149 (rG4) with the acridine orange derivative C8 , a G4 ligand stabilizer possessing anticancer activity, and the protein nucleolin (overexpressed in cancer cells) were investigated by Nuclear Magnetic Resonance (NMR) spectroscopy. For the rG4/C8 complex, the results revealed a strong stabilizing interaction between the aromatic core and the iodinated ring of the C8 ligand with the rG4 structure. The NMR study revealed also different interaction patterns between nucleolin and rG4 and nucleolin and rG4/C8 complex. In the absence of the ligand, rG4 establishes interactions with polar residues of the protein while for the rG4/C8 complex, these contacts are mainly established with amino acids that have hydrophobic side chains. However, nucleolin chemical shift perturbation studies in the presence of rG4 or rG4/C8 reveal the same location between domains 1 and 2 of the protein, which suggests that the rG4 and rG4/C8 complex bind in this region. This puzzling structural study opens a new framework to study rG4/ligand/nucleolin complexes that might impact the biogenesis of miRNA 149.

Bacteria-derived outer membrane vesicles engineered with over-expressed pre-miRNA as delivery nanocarriers for cancer therapy.

Outer membrane vesicles (OMVs) of Escherichia coli as nanoscale spherical vesicles have been recently used in cancer therapy as drug carriers. However, most of them need complicated methods to load cargos. Herein, we proposed an inexpensive and potentially mass-produced method for the preparation of OMV engineered with over-expressed pre-miRNA. In this work, we found that OMV can be released and inherit over-expressed tRNALys-pre-miRNA from mother E. coli that directly used for the tumor therapy. The eukaryotic cells infection experiments revealed that the over-expressed pre-miRNA inside OMV could be released and processed into mature miRNAs with the aid of the camouflage of "tRNA scaffold". Moreover, the group in vivo treated with targeted OMVtRNA-pre-miR-126 obviously inhibited the expression of target oncogenic CXCR4, and significantly restrain the proliferation of breast cancer tissues. Together, these findings indicated that the OMV-based platform is a versatile and powerful strategy for personalized tumor therapy directly and specificity.

Challenging Cellular Homeostasis: Spatial and Temporal Regulation of miRNAs.

Mature microRNAs (miRNAs) are single-stranded non-coding RNA (ncRNA) molecules that act in post-transcriptional regulation in animals and plants. A mature miRNA is the end product of consecutive, highly regulated processing steps of the primary miRNA transcript. Following base-paring of the mature miRNA with its mRNA target, translation is inhibited, and the targeted mRNA is degraded. There are hundreds of miRNAs in each cell that work together to regulate cellular key processes, including development, differentiation, cell cycle, apoptosis, inflammation, viral infection, and more. In this review, we present an overlooked layer of cellular regulation that addresses cell dynamics affecting miRNA accessibility. We discuss the regulation of miRNA local storage and translocation among cell compartments. The local amounts of the miRNAs and their targets dictate their actual availability, which determines the ability to fine-tune cell responses to abrupt or chronic changes. We emphasize that changes in miRNA storage and compactization occur under induced stress and changing conditions. Furthermore, we demonstrate shared principles on cell physiology, governed by miRNA under oxidative stress, tumorigenesis, viral infection, or synaptic plasticity. The evidence presented in this review article highlights the importance of spatial and temporal miRNA regulation for cell physiology. We argue that limiting the research to mature miRNAs within the cytosol undermines our understanding of the efficacy of miRNAs to regulate cell fate under stress conditions.

Bm-miR172c-5p Regulates Lignin Biosynthesis and Secondary Xylem Thickness by Altering the Ferulate 5 Hydroxylase Gene in Bacopa monnieri.

MicroRNAs (miRNAs) are small non-coding, endogenous RNAs containing 20-24 nucleotides that regulate the expression of target genes involved in various plant processes. A total of 1,429 conserved miRNAs belonging to 95 conserved miRNA families and 12 novel miRNAs were identified from Bacopa monnieri using small RNA sequencing. The Bm-miRNA target transcripts related to the secondary metabolism were further selected for validation. The Bm-miRNA expression in shoot and root tissues was negatively correlated with their target transcripts. The Bm-miRNA cleavage sites were mapped within the coding or untranslated region as depicted by the modified RLM-RACE. In the present study, we validate three miRNA targets, including asparagine synthetase, cycloartenol synthase and ferulate 5 hydroxylase (F5H) and elucidate the regulatory role of Bm-miR172c-5p, which cleaves the F5H gene involved in the lignin biosynthesis. Overexpression (OE) of Bm-miR172c-5p precursor in B. monnieri suppresses F5H gene, leading to reduced lignification and secondary xylem thickness under control and drought stress. By contrast, OE of endogenous target mimics (eTMs) showed enhanced lignification and secondary xylem thickness leading to better physiological response under drought stress. Taken together, we suggest that Bm-miRNA172c-5p might be a key player in maintaining the native phenotype of B. monnieri under control and different environmental conditions.

Molecular characterization of miRNA genes and their expression in Dimocarpus longan Lour.

MAIN CONCLUSION: A genome-wide analysis of longan miRNA genes was conducted, and full-length pri-miRNA transcripts were cloned. Bioinformatics and expression analyses contributed to the functional characterization of longan miRNA genes. MicroRNAs are important for the post-transcriptional regulation of target genes. However, little is known about the transcription and regulation of miRNA genes in longan (Dimocarpus longan Lour.). In this study, 80 miRNA precursors (pre-miRNA) were predicted, and their secondary structure, size, conservation, and diversity were analyzed. Furthermore, the full-length cDNA sequences of 13 longan primary miRNAs (pri-miRNAs) were amplified by RLM-RACE and SMART-RACE and analyzed, which revealed that longan pri-miRNA transcripts have multiple transcription start sites (TSSs) and the downstream pre-miRNAs are polymorphic. Accordingly, the longan pri-miRNAs and protein-encoding genes may have similar transcriptional specificities. An analysis of the longan miRNA gene promoter elements indicated that the three most abundant cis-acting elements were light-responsive, stress-responsive, and hormone-responsive elements. A quantitative real-time PCR assay elucidated the potential spatial and temporal expression patterns of longan pre-miRNAs during the early stages of somatic embryogenesis (SE) and in different longan organs/tissues. This is the first report regarding the molecular characterization of miRNA genes and their expression profiles in longan. The generated data may serve as a foundation for future research aimed at clarifying the longan miRNA gene functions.

Molecular characterization of DICER1-mutated pituitary blastoma.

Pituitary blastoma (PitB) has recently been identified as a rare and potentially lethal pediatric intracranial tumor. All cases that have been studied molecularly possess at least one DICER1 pathogenic variant. Here, we characterized nine pituitary samples, including three fresh frozen PitBs, three normal fetal pituitary glands and three normal postnatal pituitary glands using small-RNA-Seq, RNA-Seq, methylation profiling, whole genome sequencing and Nanostring® miRNA analyses; an extended series of 21 pituitary samples was used for validation purposes. These analyses demonstrated that DICER1 RNase IIIb hotspot mutations in PitBs induced improper processing of miRNA precursors, resulting in aberrant 5p-derived miRNA products and a skewed distribution of miRNAs favoring mature 3p over 5p miRNAs. This led to dysregulation of hundreds of 5p and 3p miRNAs and concomitant dysregulation of numerous mRNA targets. Gene expression analysis revealed PRAME as the most significantly upregulated gene (500-fold increase). PRAME is a member of the Retinoic Acid Receptor (RAR) signaling pathway and in PitBs, the RAR, WNT and NOTCH pathways are dysregulated. Cancer Hallmarks analysis showed that PI3K pathway is activated in the tumors. Whole genome sequencing demonstrated a quiet genome with very few somatic alterations. The comparison of methylation profiles to publicly available data from ~ 3000 other central nervous system tumors revealed that PitBs have a distinct methylation profile compared to all other tumors, including pituitary adenomas. In conclusion, this comprehensive characterization of DICER1-related PitB revealed key molecular underpinnings of PitB and identified pathways that could potentially be exploited in the treatment of this tumor.

Selectivity of Exportin 5 binding to human precursor microRNAs.

Exportin 5 (Exp5, XPO5) is a nuclear export factor that functions in the microRNA (miRNA) biogenesis pathway to transport precursor miRNAs (pre-miRNAs) from the nucleus to the cytoplasm. Most of our current understanding of the Exp5 and pre-miRNA interaction is based on the investigation of how Exp5 binds to human pre-miR-30a (pre-miR-30 for short). As there are hundreds of human miRNA genes, how representative pre-miR-30 is, whether or how Exp5 interacts with distinct cargoes differentially, or whether Exp5 regulates miRNA expression, is unknown. Here we examined and compared the interactions between Exp5 and 157 human pre-miRNAs. We found that Exp5 binds distinct pre-miRNAs with modest variations in efficiencies, with the 3' overhang and the apical loop in pre-miRNAs being the two major discriminating factors. Exp5 binding efficiencies do not significantly correlate with endogenous miRNA expression, suggesting that Exp5 activity does not contribute to differential miRNA expression in vivo. Nonetheless, in human cells with reduced Exp5 levels, preferential Exp5 binding correlated with miRNA expression changes. Thus, our study provides a global picture of how Exp5 interacts with human pre-miRNAs and its role in regulating miRNA expression.Abbreviations: Exp5: Exportin 5; miRNA: microRNA; pri-miRNA: primary microRNA; pre-miRNA: precursor microRNA; nt: nucleotide.

Development and Modification of Pre-miRNAs with a FRET Dye Pair for the Intracellular Visualization of Processing Intermediates That Are Generated in Cells.

microRNAs (miRNAs) are small non-coding ribonucleic acids (RNAs), which regulate gene expression via the RNA interference (RNAi) system. miRNAs have attracted enormous interest because of their biological significance and disease relationship. In cell systems, the generation of miRNA is regulated by multiple steps: the transfer of primary miRNA from the nucleus to the cytosol, the generation of the precursor-miRNA (pre-miRNA), the production of double-stranded RNA from pre-miRNA by the Dicer, the interaction with protein argonaute-2 (AGO2), and the subsequent release of one strand to form miRISC with AGO2. In this study, we attempt to visualize the intermediates that were generated in the miRNA-maturation step in the cells to acquire a detailed understanding of the maturation process of miRNA. To achieve this, we developed pre-miRNAs labeling with a Dicer- or AGO2-responsible fluorescence resonance energy transfer (FRET) dye pair. We observed that modifications with the dye at suitable positions did not interfere with the biological activities of pre-miRNAs. Further, imaging analyses employing these pre-miRNAs demonstrated that the processing of pre-miRNA promoted the accumulation of miRNA at the specific foci in the cytosol. The FRET-labeled pre-miRNA would further elucidate the mechanisms of the RNAi process and provide the basis for development of nucleic acid drugs working in the RNAi system.

Complex Conformational Dynamics of the Heart Failure-Associated Pre-miRNA-377 Hairpin Revealed by Single-Molecule Optical Tweezers.

Pre-miRNA-377 is a hairpin-shaped regulatory RNA associated with heart failure. Here, we use single-molecule optical tweezers to unzip pre-miRNA-377 and study its stability and dynamics. We show that magnesium ions have a strong stabilizing effect, and that sodium ions stabilize the hairpin more than potassium ions. The hairpin unfolds in a single step, regardless of buffer composition. Interestingly, hairpin folding occurs either in a single step (type 1) or through the formation of intermediates, in multiple steps (type 2) or gradually (type 3). Type 3 occurs only in the presence of both sodium and magnesium, while type 1 and 2 take place in all buffers, with type 1 being the most prevalent. By reducing the size of the native hairpin loop from fourteen to four nucleotides, we demonstrate that the folding heterogeneity originates from the large size of the hairpin loop. Further, while efficient pre-miRNA-377 binders are lacking, we demonstrate that the recently developed C2 ligand displays bimodal activity: it enhances the mechanical stability of the pre-miRNA-377 hairpin and perturbs its folding. The knowledge regarding pre-miRNA stability and dynamics that we provide is important in understanding its regulatory function and how it can be modulated to achieve a therapeutic effect, e.g., in heart failure treatment.

The RNA-binding protein SART3 promotes miR-34a biogenesis and G1 cell cycle arrest in lung cancer cells.

MicroRNAs (miRNAs or miRs) are small, noncoding RNAs that are implicated in the regulation of most biological processes. Global miRNA biogenesis is altered in many cancers, and RNA-binding proteins play a role in miRNA biogenesis, presenting a promising avenue for targeting miRNA dysregulation in diseases. miR-34a exhibits tumor-suppressive activities by targeting cell cycle regulators CDK4/6 and anti-apoptotic factor BCL-2, among other regulatory pathways such as Wnt, TGF-ß, and Notch signaling. Many cancers exhibit down-regulation or loss of miR-34a, and synthetic miR-34a supplementation has been shown to inhibit tumor growth in vivo However, the post-transcriptional mechanisms that cause miR-34a loss in cancer are not entirely understood. Here, using a proteomics-mediated approach in non-small-cell lung cancer (NSCLC) cells, we identified squamous cell carcinoma antigen recognized by T-cells 3 (SART3) as a putative pre-miR-34a-binding protein. SART3 is a spliceosome recycling factor and nuclear RNA-binding protein with no previously reported role in miRNA regulation. We found that SART3 binds pre-miR-34a with higher specificity than pre-let-7d (used as a negative control) and elucidated a new functional role for SART3 in NSCLC cells. SART3 overexpression increased miR-34a levels, down-regulated the miR-34a target genes CDK4/6, and caused a cell cycle arrest in the G1 phase. In vitro binding experiments revealed that the RNA-recognition motifs within the SART3 sequence are responsible for selective pre-miR-34a binding. Our results provide evidence for a significant role of SART3 in miR-34a biogenesis and cell cycle progression in NSCLC cells.

The dynamic recruitment of TRBP to neuronal membranes mediates dendritogenesis during development.

MicroRNAs are important regulators of local protein synthesis during neuronal development. We investigated the dynamic regulation of microRNA production and found that the majority of the microRNA-generating complex, consisting of Dicer, TRBP, and PACT, specifically associates with intracellular membranes in developing neurons. Stimulation with brain-derived neurotrophic factor (BDNF), which promotes dendritogenesis, caused the redistribution of TRBP from the endoplasmic reticulum into the cytoplasm, and its dissociation from Dicer, in a Ca2+-dependent manner. As a result, the processing of a subset of neuronal precursor microRNAs, among them the dendritically localized pre-miR16, was impaired. Decreased production of miR-16-5p, which targeted the BDNF mRNA itself, was rescued by expression of a membrane-targeted TRBP Moreover, miR-16-5p or membrane-targeted TRBP expression blocked BDNF-induced dendritogenesis, demonstrating the importance of neuronal TRBP dynamics for activity-dependent neuronal development. We propose that neurons employ specialized mechanisms to modulate local gene expression in dendrites, via the dynamic regulation of microRNA biogenesis factors at intracellular membranes of the endoplasmic reticulum, which in turn is crucial for neuronal dendrite complexity and therefore neuronal circuit formation and function.

Characterization of a G-Quadruplex Structure in Pre-miRNA-1229 and in Its Alzheimer's Disease-Associated Variant rs2291418: Implications for miRNA-1229 Maturation.

Alzheimer's disease (AD), the most common age-related neurodegenerative disease, is associated with various forms of cognitive and functional impairment that worsen with disease progression. AD is typically characterized as a protein misfolding disease, in which abnormal plaques form due to accumulation of tau and ß-amyloid (Aß) proteins. An assortment of proteins is responsible for the processing and trafficking of Aß, including sortilin-related receptor 1 (SORL1). Recently, a genome-wide association study of microRNA-related variants found that a single nucleotide polymorphism (SNP) rs2291418 within premature microRNA-1229 (pre-miRNA-1229) is significantly associated with AD. Moreover, the levels of the mature miRNA-1229-3p, which has been shown to regulate the SORL1 translation, are increased in the rs2291418 pre-miRNA-1229 variant. In this study we used various biophysical techniques to show that pre-miRNA-1229 forms a G-quadruplex secondary structure that coexists in equilibrium with the canonical hairpin structure, potentially controlling the production of the mature miR-1229-3p, and furthermore, that the AD-associated SNP rs2291418 pre-miR-1229 changes the equilibrium between these structures. Thus, the G-quadruplex structure we identified within pre-miRNA-1229 could potentially act as a novel therapeutic target in AD.

Cloning, expression, and characterization of the zebrafish Dicer and Drosha enzymes.

The biogenesis of animal microRNAs (miRNAs) involves transcription followed by a series of processing steps, with Drosha and Dicer being two key enzymes that cleave primary miRNA (pri-miRNA) and precursor miRNA (pre-miRNA) transcripts, respectively. While human and fly Dicer and human Drosha are well studied, their homologs in other organisms have not been biochemically characterized, leaving open the question of whether their miRNA substrate specificities and regulatory functions are conserved. Zebrafish is a widely used model organism, but its miRNA processing enzymes have never been reconstituted and analyzed. In this study we cloned and constructed expression plasmids encoding zebrafish Dicer, Drosha, and their accessory proteins TARBP2 and DGCR8. After transfection of human cell cultures, we isolated the recombinant protein complexes. We found that zebrafish Dicer bound TARBP2, but Dicer alone exhibited significant pre-miRNA processing activity. On the other hand, zebrafish Drosha associated with DGCR8, and both were required to cleave pri-miRNAs. The Drosha/DGCR8 holoenzyme preferred pri-miRNAs with a large terminal loop, an extended duplex region, and flanking single stranded RNAs. These results lay the foundation for future studies of the regulatory roles and conserved mechanisms of Drosha and Dicer.

Inhibition of pre-miRNA-136 processing by Dicer with small molecule BzDANP suggested the formation of ternary complex of pre-miR-136-BzDANP-Dicer.

Small-molecule modulators, along with antisense oligonucleotide, would be powerful tools and potential drug candidates for modulating miRNA-related gene expressions. The mechanism of the inhibitory effect of the C-bulge binding small molecule BzDANP for the Dicer processing reaction of pre-miR-136 was discussed on the data obtained by SPR, NMR, and kinetic analysis for Dicer processing. SPR and NMR analysis showed the preference of BzDANP binding to the C-bulge. Michaelis-Menten analysis suggested the formation of a ternary complex pre-miR-136-BzDANP-Dicer during the Dicer-cleavage reaction of pre-miR-136 in the presence of BzDANP. The inhibitory effect of BzDANP is likely attributed to the slower reaction from the ternary complex than that from the binary pre-miR-136-Dicer complex.

Dysregulation of microRNA biogenesis machinery in cancer.

MicroRNAs (miRNAs) are integral to the gene regulatory network. A single miRNA is capable of controlling the expression of hundreds of protein coding genes and modulate a wide spectrum of biological functions, such as proliferation, differentiation, stress responses, DNA repair, cell adhesion, motility, inflammation, cell survival, senescence and apoptosis, all of which are fundamental to tumorigenesis. Overexpression, genetic amplification, and gain-of-function mutation of oncogenic miRNAs ("onco-miRs") as well as genetic deletion and loss-of-function mutation of tumor suppressor miRNAs ("suppressor-miRs") are linked to human cancer. In addition to the dysregulation of a specific onco-miR or suppressor-miRs, changes in global miRNA levels resulting from a defective miRNA biogenesis pathway play a role in tumorigenesis. The function of individual onco-miRs and suppressor-miRs and their target genes in cancer has been described in many different articles elsewhere. In this review, we primarily focus on the recent development regarding the dysregulation of the miRNA biogenesis pathway and its contribution to cancer.

High-yield production of human Dicer by transfection of human HEK293-EBNA1 cells grown in suspension.

BACKGROUND: Dicer is a 219-kDa protein that plays key roles in gene regulation, particularly as the ribonuclease III enzyme responsible for cleaving precursor miRNA substrates. Its enzymatic activity is highly regulated by protein factors, and this regulation can impact on the levels of miRNAs and modulate the behavior of a cell. To better understand the underlying mechanisms of regulation, detailed enzymatic and structural characterization of Dicer are needed. However, these types of studies generally require several milligrams of recombinant protein, and efficient preparation of such quantities of pure human Dicer remains a challenge. To prepare large quantities of human Dicer, we have optimized transfection in HEK293-6E cells grown in suspension and streamlined a purification procedure. RESULTS: Transfection conditions were first optimized to achieve expression levels between 10 and 18 mg of recombinant Dicer per liter of culture. A three-step purification protocol was then developed that yields 4-9 mg of purified Dicer per liter of culture in a single day. From SEC-MALS/RI analysis and negative stain TEM, we confirmed that the purified protein is monomerically pure ( ≥ 98%) and folds with the characteristic L-shape geometry. Using an electrophoretic mobility shift assay, a dissociation constant (Kd) of 5 nM was measured for Dicer binding to pre-let-7a-1, in agreement with previous reports. However, when probing the cleavage activity of Dicer for pre-let-7a-1, we measured kcat (7.2 ± 0.5 min- 1) and KM (1.2 ± 0.3 µM) values that are much higher than previously reported due to experimental conditions that better respect the steady-state assumption. CONCLUSIONS: The expression and purification protocols described here provide high yields of monomerically pure and active human Dicer. Cleavage studies of a pre-let-7 substrate with this purified Dicer reveal higher kcat and KM values than previously reported and support the current view that conformational changes are associated with substrate binding. Large quantities of highly pure Dicer will be valuable for future biochemical, biophysical and structural investigations of this key protein of the miRNA pathway.

ARiBo pull-down for riboproteomic studies based on label-free quantitative mass spectrometry.

As part of their normal life cycle, most RNA molecules associate with several proteins that direct their fate and regulate their function. Here, we describe a novel method for identifying proteins that associate with a target RNA. The procedure is based on the ARiBo method for affinity purification of RNA, which was originally developed to quickly purify RNA with high yields and purity under native conditions. The ARiBo method was further optimized using in vitro transcribed RNA to capture RNA-associating proteins from cellular extracts with high yields and low background protein contamination. For these RNA pull-downs, stem-loops present in the immature forms of let-7 miRNAs (miRNA stem-loops) were used as the target RNAs. Label-free quantitative mass spectrometry analysis allowed for the reliable identification of proteins that are specific to the stem-loops present in the immature forms of two miRNAs, let-7a-1 and let-7g. Several proteins known to bind immature forms of these let-7 miRNAs were identified, but with an improved coverage compared to previous studies. In addition, several novel proteins were identified that better define the protein interactome of the let-7 miRNA stem-loops and further link let-7 biogenesis to important biological processes such as development and tumorigenesis. Thus, combining the ARiBo pull-down method with label-free quantitative mass spectrometry provides an effective proteomic approach for identification of proteins that associate with a target RNA.