Cutoff Suppresses RNA Polymerase II Termination to Ensure Expression of piRNA Precursors.

Small non-coding RNAs called piRNAs serve as guides for an adaptable immune system that represses transposable elements in germ cells of Metazoa. In Drosophila the RDC complex, composed of Rhino, Deadlock and Cutoff (Cuff) bind chromatin of dual-strand piRNA clusters, special genomic regions, which encode piRNA precursors. The RDC complex is required for transcription of piRNA precursors, though the mechanism by which it licenses transcription remained unknown. Here, we show that Cuff prevents premature termination of RNA polymerase II. Cuff prevents cleavage of nascent RNA at poly(A) sites by interfering with recruitment of the cleavage and polyadenylation specificity factor (CPSF) complex. Cuff also protects processed transcripts from degradation by the exonuclease Rat1. Our work reveals a conceptually different mechanism of transcriptional enhancement. In contrast to other factors that regulate termination by binding to specific signals on nascent RNA, the RDC complex inhibits termination in a chromatin-dependent and sequence-independent manner.

Retinoic Acid Upregulates METTL14 Expression and the m6A Modification Level to Inhibit the Proliferation of Embryonic Palate Mesenchymal Cells in Cleft Palate Mice.

Cleft palate only (CPO) is one of the most common craniofacial birth defects. Environmental factors can induce cleft palate by affecting epigenetic modifications such as DNA methylation, histone acetylation, and non-coding RNA. However, there are few reports focusing on the RNA modifications. In this study, all-trans retinoic acid (atRA) was used to simulate environmental factors to induce a C57BL/6J fetal mouse cleft palate model. Techniques such as dot blotting and immunofluorescence were used to find the changes in m6A modification when cleft palate occurs. RNA-seq and KEGG analysis were used to screen for significantly differentially expressed pathways downstream. Primary mouse embryonic palate mesenchymal (MEPM) cells were successfully isolated and used for in vitro experimental verification. We found that an increased m6A methylation level was correlated with suppressed cell proliferation in the palatine process mesenchyme of cleft palate mice. This change is due to the abnormally high expression of m6A methyltransferase METTL14. When using siRNAs and the m6A methyltransferase complex inhibitor SAH to interfere with the expression or function of METTL14, the teratogenic effect of atRA on primary cells was partially alleviated. In conclusion, METTL14 regulates palatal mesenchymal cell proliferation and cycle-related protein expression relies on m6A methylation modification, affecting the occurrence of cleft palate.

Hydrogel delivery of purinergic enzymes improves cardiac ischemia/reperfusion injury.

RATIONALE: The innate immune response contributes to cardiac injury in myocardial ischemia/reperfusion (MI/R). Neutrophils are an important early part of the innate immune response to MI/R. Adenosine, an endogenous purine, is a known innate immune modulator and inhibitor of neutrophil activation. However, its delivery to the heart is limited by its short half-life (<30 s) and off-target side effects. CD39 and CD73 are anti-inflammatory homeostatic enzymes that can generate adenosine from phosphorylated adenosine substrate such as ATP released from injured tissue. OBJECTIVE: We hypothesize that hydrogel-delivered CD39 and CD73 target the local early innate immune response, reduce neutrophil activation, and preserve cardiac function in MI/R injury. METHODS AND RESULTS: We engineered a poly(ethylene) glycol (PEG) hydrogel loaded with the adenosine-generating enzymes CD39 and CD73. We incubated the hydrogels with neutrophils in vitro and showed a reduction in hydrogen peroxide production using Amplex Red. We demonstrated availability of substrate for the enzymes in the myocardium in MI/R by LC/MS, and tested release kinetics from the hydrogel. On echocardiography, global longitudinal strain (GLS) was preserved in MI/R hearts treated with the loaded hydrogel. Delivery of purinergic enzymes via this synthetic hydrogel resulted in lower innate immune infiltration into the myocardium post-MI/R, decreased markers of macrophage and neutrophil activation (NETosis), and decreased leukocyte-platelet complexes in circulation. CONCLUSIONS: In a rat model of MI/R injury, CD39 and CD73 delivered via a hydrogel preserve cardiac function by modulating the innate immune response.

N6-Methyladenosine Reader YTHDF1 Promotes ARHGEF2 Translation and RhoA Signaling in Colorectal Cancer.

BACKGROUND & AIMS: N6-methyladenosine (m6A) governs the fate of RNAs through m6A readers. Colorectal cancer (CRC) exhibits aberrant m6A modifications and expression of m6A regulators. However, how m6A readers interpret oncogenic m6A methylome to promote malignant transformation remains to be illustrated. METHODS: YTH N6-methyladenosine RNA binding protein 1 (Ythdf1) knockout mouse was generated to determine the effect of Ythdf1 in CRC tumorigenesis in vivo. Multiomic analysis of RNA-sequencing, m6A methylated RNA immunoprecipitation sequencing, YTHDF1 RNA immunoprecipitation sequencing, and proteomics were performed to unravel targets of YTHDF1 in CRC. The therapeutic potential of targeting YTHDF1-m6A-Rho/Rac guanine nucleotide exchange factor 2 (ARHGEF2) was evaluated using small interfering RNA (siRNA) encapsulated by lipid nanoparticles (LNP). RESULTS: DNA copy number gain of YTHDF1 is a frequent event in CRC and contributes to its overexpression. High expression of YTHDF1 is significantly associated with metastatic gene signature in patient tumors. Ythdf1 knockout in mice dampened tumor growth in an inflammatory CRC model. YTHDF1 promotes cell growth in CRC cell lines and primary organoids and lung and liver metastasis in vivo. Integrative multiomics analysis identified RhoA activator ARHGEF2 as a key downstream target of YTHDF1. YTHDF1 binds to m6A sites of ARHGEF2 messenger RNA, resulting in enhanced translation of ARHGEF2. Ectopic expression of ARHGEF2 restored impaired RhoA signaling, cell growth, and metastatic ability both in vitro and in vivo caused by YTHDF1 loss, verifying that ARHGEF2 is a key target of YTHDF1. Finally, ARHGEF2 siRNA delivered by LNP significantly suppressed tumor growth and metastasis in vivo. CONCLUSIONS: We identify a novel oncogenic epitranscriptome axis of YTHDF1-m6A-ARHGEF2, which regulates CRC tumorigenesis and metastasis. siRNA-delivering LNP drug validated the therapeutic potential of targeting this axis in CRC.

Selective regulation of human TRAAK channels by biologically active phospholipids.

TRAAK is an ion channel from the two-pore domain potassium (K2P) channel family with roles in maintaining the resting membrane potential and fast action potential conduction. Regulated by a wide range of physical and chemical stimuli, the affinity and selectivity of K2P4.1 toward lipids remains poorly understood. Here we show the two isoforms of K2P4.1 have distinct binding preferences for lipids dependent on acyl chain length and position on the glycerol backbone. The channel can also discriminate the fatty acid linkage at the SN1 position. Of the 33 lipids interrogated using native mass spectrometry, phosphatidic acid had the lowest equilibrium dissociation constants for both isoforms of K2P4.1. Liposome potassium flux assays with K2P4.1 reconstituted in defined lipid environments show that those containing phosphatidic acid activate the channel in a dose-dependent fashion. Our results begin to define the molecular requirements for the specific binding of lipids to K2P4.1.

Synthetic Lethality by Co-Inhibition of Androgen Receptor and Polyadenosine Diphosphate-Ribose in Metastatic Prostate Cancer.

Androgen receptor pathway inhibitors (ARPI) and polyadenosine diphosphate-ribose inhibitors (PARPi) are part of the standard of care in patients with metastatic castration-resistant prostate cancer (mCRPC). There is biological evidence that the association of ARPI and PARPi could have a synergistic effect; therefore, several ongoing clinical trials are investigating the efficacy of this combination with preliminary results that are not perfectly concordant in identifying patients who can obtain the most benefit from this therapeutic option. The purpose of this review is to describe the PARPi mechanisms of action and to analyze the biological mechanisms behind the interplay between the androgen receptor and the PARPi system to better understand the rationale of the ARPI + PARPi combinations. Furthermore, we will summarize the preliminary results of the ongoing studies on these combinations, trying to understand in which patients to apply. Finally, we will discuss the clinical implications of this combination and its possible future perspectives.

General Label-Free Fluorescent Aptamer Binding Assay Using Cationic Conjugated Polymers.

With more and more new aptamers being reported, a general, cost-effective yet reliable aptamer binding assay is still needed. Herein, we studied cationic conjugated polymer (CCP)-based binding assays taking advantage of the conformational change of aptamer after binding with a target, which is reflected by the fluorescence change of the CCP. Poly(3-(3'-N,N,N-triethylamino-1'-propyloxy)-4-methyl-2,5-thiophene hydrochloride) (PMNT) was used as a model CCP in this study, and the optimal buffer was close to physiological conditions with 100 mM NaCl and 10 mM MgCl2. We characterized four aptamers for K+, adenosine, cortisol, and caffeine. For cortisol and caffeine, the drop in the 580 nm peak intensity was used for quantification, whereas for K+ and adenosine, the fluorescence ratio at 580 over 530 nm was used. The longer stem of the stem-loop structured aptamer facilitated binding of the target and enlarged the detection signal. High specificity was achieved in differentiating targets with analogues. Compared with the SYBR Green I dye-based staining method, our method achieved equal or even higher sensitivity. Therefore, this assay is practicable as a general aptamer binding assay. The simple, label-free, quick response, and cost-effective features will make it a useful method to evaluate aptamer binding. At the same time, this system can also serve as label-free biosensors for target detection.

In Situ Measurement of ATP in Single Cells by an Amphiphilic Aptamer-Assisted Electrochemical Nano-Biosensor.

In situ and quantitative measurements of adenosine 5'-triphosphate (ATP) in single living cells are highly desired for understanding several sorts of necessary physiological and pathological processes. Due to its small size and high sensitivity, an ultra-microelectrode can be used for single-cell analysis. However, ATP is difficult to detect in single cells because it is nonelectroactive and low in content. Herein, we introduced an electrochemical nano-biosensor based on an amphiphilic aptamer-assisted carbon fiber nanoelectrode (aptCFNE) with high signal-to-noise ratio. The low current (e.g., 60 pA) and the tiny diameter of the tip (ca. 400 nm) of the nanosensor made it noninvasive to living cells. The amphiphilic aptamer has good biocompatibility and can be stably modified to the surface of functionalized electrodes. CFNE, which was modified with ferrocene-labeled aptamer, could quickly and selectively detect ATP content in the nucleus, cytoplasm, and extracellular space of single HeLa cells. The results showed that the ATP contents in the nucleus, cytoplasm, and extracellular space were 568 ± 9, 461 ± 20, and 312 ± 4 µM, respectively. The anticancer drug treatment effects on the cellular level were further recorded, which was of great significance for understanding ATP-related biological processes and drug screenings. This strategy is universally applicable to detect other targets by changing the aptamer sequence, which will greatly improve our understanding of cell heterogeneity and provide a more reliable scientific basis for exploring major diseases at the single-cell level.

Stage-specific requirement for METTL3-dependent m6A modification during dental pulp stem cell differentiation.

BACKGROUND: N6-methyladenosine (m6A) is the most prevalent epigenetic modification in eukaryotic messenger RNAs and plays a critical role in cell fate transition. However, it remains to be elucidated how m6A marks functionally impact the transcriptional cascades that orchestrate stem cell differentiation. The present study focuses on the biological function and mechanism of m6A methylation in dental pulp stem cell (DPSC) differentiation. METHODS: m6A RNA immunoprecipitation sequencing was utilized to assess the m6A-mRNA landscape during DPSC differentiation. Ectopic transplantation of DPSCs in immunodeficient mice was conducted to verify the in vitro findings. RNA sequencing and m6A RNA immunoprecipitation sequencing were combined to identify the candidate targets. RNA immunoprecipitation and RNA/protein stability of Noggin (NOG) were evaluated. The alteration in poly(A) tail was measured by 3'-RACE and poly(A) tail length assays. RESULTS: We characterized a dynamic m6A-mRNA landscape during DPSC mineralization with increasing enrichment in the 3' untranslated region (UTR). Methyltransferase-like 3 (METTL3) was identified as the key m6A player, and METTL3 knockdown disrupted functional DPSC differentiation. Moreover, METTL3 overexpression enhanced DPSC mineralization. Increasing m6A deposition in the 3' UTR restricted NOG expression, which is required for DPSC mineralization. This stage-specific m6A methylation and destabilization of NOG was suppressed by METTL3 knockdown only in differentiated DPSCs. Furthermore, METTL3 promotes the degradation of m6A-tagged NOG by shortening the poly(A) tail length in the differentiated stage. CONCLUSIONS: Our results address an essential role of dynamic m6A signaling in the temporal control of DPSC differentiation and provide new insight into epitranscriptomic mechanisms in stem cell-based therapy.

Human dental pulp cells modulate CD8+ T cell proliferation and efficiently degrade extracellular ATP to adenosine in vitro.

The pulp of human teeth contains a population of self-renewing stem cells that can regulate the functions of immune cells. When applied to patients, these cells can protect tissues from damage by excessive inflammation. We confirm that dental pulp cells effectively inhibit the proliferation and activation of cytotoxic T cells in vitro, and show that they carry high levels of CD73, a key enzyme in the conversion of pro-inflammatory extracellular ATP to immunosuppressive adenosine. Given their accessibility and abundance, as well as their potential for allogeneic administration, dental pulp cells provide a valuable source for immunomodulatory therapy.

A Hydrogel Strategy to Augment Tissue Adenosine to Improve Hindlimb Perfusion.

[Figure: see text].

Synthesis of Nucleoside-like Molecules from a Pyrolysis Product of Cellulose and Their Computational Prediction as Potential SARS-CoV-2 RNA-Dependent RNA Polymerase Inhibitors.

(1R,5S)-1-Hydroxy-3,6-dioxa-bicyclo[3.2.1]octan-2-one, available by an efficient catalytic pyrolysis of cellulose, has been applied as a chiral building block in the synthesis of seven new nucleoside analogues, with structural modifications on the nucleobase moiety and on the carboxyl- derived unit. The inverted configuration by Mitsunobu reaction used in their synthesis was verified by 2D-NOESY correlations, supported by the optimized structure employing the DFT methods. An in silico screening of these compounds as inhibitors of SARS-CoV-2 RNA-dependent RNA polymerase has been carried out in comparison with both remdesivir, a mono-phosphoramidate prodrug recently approved for COVID-19 treatment, and its ribonucleoside metabolite GS-441524. Drug-likeness prediction and data by docking calculation indicated compound 6 [=(3S,5S)-methyl 5-(hydroxymethyl)-3-(6-(4-methylpiperazin-1-yl)-9H-purin-9-yl)tetrahydrofuran-3-carboxylate] as the best candidate. Furthermore, molecular dynamics simulation showed a stable interaction of structure 6 in RNA-dependent RNA polymerase (RdRp) complex and a lower average atomic fluctuation than GS-441524, suggesting a well accommodation in the RdRp binding pocket.

Adenosine A2A Receptor Antagonist Improves Cognitive Impairment by Inhibiting Neuroinflammation and Excitatory Neurotoxicity in Chronic Periodontitis Mice.

The adenosine A2A receptor antagonist SCH58261 has been reported to have anti-inflammatory effects. However, its role in chronic periodontitis (CP)-induced cognitive impairment, which is associated with Porphyromonas gingivalis lipopolysaccharide (P. gingivalis LPS), remains unclear. This study investigated the role of SCH58261 in mice with CP-induced cognitive impairment. C57BL/6J mice were used to develop CP model by injecting 0.5 mg/kg P. gingivalis LPS into the palatal gingival sulcus of maxillary first molars twice a week for four weeks. The mice were divided into control, P. gingivalis LPS (P-LPS), P-LPS + SCH58261, and SCH58261 groups. The passive avoidance test (PAT) and Morris water maze (MWM) were used to assess cognition in mice. Furthermore, CD73/adenosine, neuroinflammation, glutamate transporters, and glutamate were assessed. Compared with the P-LPS group, 0.1 and 0.5 mg/kg SCH58261 increased latency and decreased error times in PAT, but increased platform crossing number in MWM. SCH58261 inhibited microglial activation, and decreased pro-inflammatory cytokines and glutamate levels, but increased GLT-1 and PSD95 expression in the hippocampus. This was the first report of SCH58261 treatment for CP-induced cognitive impairment, which may be related to its anti-inflammatory activities and anti-glutamate excitatory neurotoxicity. This suggests that SCH58261 can be used as a novel agent to treat cognitive impairment.

m6A regulator-mediated RNA methylation modification patterns are involved in immune microenvironment regulation of periodontitis.

The role of epigenetic regulation in immunity is emerging, especially for RNA N6-methyladenosine (m6A) modification. However, little is known about the role of m6A in the regulation of the immune microenvironment of periodontitis. Thus, we aim to investigate the impact of m6A modification in periodontitis immune microenvironment. The RNA modification patterns mediated by 23 m6A-regulators were systematically evaluated in 310 periodontitis samples. The impact of m6A modification on immune microenvironment characteristics was explored, including infiltrating immunocytes, immune reaction gene-sets and HLAs (human leukocyte antigen) gene. m6A phenotype-related immune genes were also identified. 17 m6A regulators were dysregulated and a 15-m6A regulator signature can well distinguish periodontitis and control samples. ALKBH5 and FMR1 are closely related to infiltrating monocyte abundance. ELAVL1 and CBLL1 are significant regulators in immune reaction of TNF_Family_Members_Receptors and Cytokine. The expression of HLA-B and HLA-DOA is affected by ALKBH5 and LRPPRC. 3 distinct RNA modification patterns mediated by 23 m6A regulators were identified. They differ from immunocyte abundance, immune reaction and HLA gene. 1631 m6A phenotype-related genes and 70 m6A-mediated immune genes were identified, and the biological functions of these were explored. Our finding demonstrated the m6A modification plays a crucial role in the diversity and complexity of the immune microenvironment of periodontitis.

In situ label-free and sensitive detection assay for cell apoptosis via polyadenosine-coralyne fluorescence enhancement strategy.

Cell apoptosis detection is vital for biological analysis and clinical application; some detection assays are already commercially available. However, it is still far from perfect and needs further improvement for less cost, time-consuming and operation demanding. TUNEL, a high market share cell apoptosis assay, depends on adulteration fluorescent labelling dUTP by terminal deoxynucleotidyl transferase(TdT) which randomly adds deoxyribonucleoside triphosphates (dNTPs) at the 3'-OH terminal of ssDNA with a template-free manner. Based on our previous work, we adopted a label-free strategy to reduce the cost and operation maintenance of TUNEL and developed a facile, rapid, convenient and in-situ assay for cell apoptosis.

Reversing Hypoxia with PLGA-Encapsulated Manganese Dioxide Nanoparticles Improves Natural Killer Cell Response to Tumor Spheroids.

The adoptive transfer of natural killer (NK) cells, which can recognize and obliterate cancer cells, provides a practical alternative to current treatment modalities to improve cancer patients' survival. However, translating NK cell therapies to treat solid tumors has proven challenging due to the tumor microenvironment (TME). Hypoxia in the TME induces immunosuppression that inhibits the cytotoxic function of NK cells. Thus, reversing hypoxia-induced immunosuppression is critical for effective adoptive NK cell immunotherapy. In this study, we use manganese dioxide nanoparticles (MnO2 NPs) to catalyze the degradation of tumor-produced hydrogen peroxide, thereby generating oxygen. For improved biocompatibility and modulation of oxygen production, the MnO2 NPs were encapsulated into poly(lactic-co-glycolic) to produce particles that are 116 nm in size and with a ζ-potential of +17 mV (PLGA-MnO2 NPs). The PLGA-MnO2 NPs showed first-order oxygen production and sustained high oxygen tension compared to equivalent amounts of bare MnO2 NPs in the presence of H2O2. The PLGA-MnO2 NPs were biocompatible, reduced hypoxia after penetration into the core of cancer spheroids, and decreased hypoxia-induced factor 1 α expression. Reducing hypoxia in the spheroid resulted in a decrease in the potent immunosuppressors, adenosine, and lactate, which was confirmed by electrospray ionization mass spectroscopy (ESI-MS). ESI-MS also showed a change in the metabolism of the amino acids aspartate, glutamine, and glutamate after hypoxia reduction in the cancer cells. Notably, the spheroids' microenvironment changes enhanced NK cells' cytotoxicity, which obliterated the spheroids. These results demonstrate that reducing hypoxia-induced immunosuppression in tumors is a potent strategy to increase the potency of cytotoxic immune cells in the TME. The developed NPs are promising new tools to improve adoptive NK cell therapy.

Multiplexing neurochemical detection with carbon fiber multielectrode arrays using fast-scan cyclic voltammetry.

Carbon fiber microelectrodes (CFMEs) have been extensively used to measure neurotransmitters with fast-scan cyclic voltammetry (FSCV) due to their ability to adsorb cationic monoamine neurotransmitters. Although FSCV, in tandem with CFMEs, provides high temporal and spatial resolution, only single-channel potentiostats and electrodes have been primarily utilized. More recently, the need and use of carbon fiber multielectrode arrays has risen to target multiple brain regions. Previous studies have shown the ability to detect dopamine using multielectrode arrays; however, they are not readily available to the scientific community. In this work, we interfaced a carbon fiber multielectrode array (MEA or multielectrode array), to a commercially available four-channel potentiostat for multiplexing neurochemical measurements. The MEA's relative performance was compared to single CFMEs where dopamine detection was found to be adsorption controlled to the electrode's surface. Multiple waveforms were applied to each fiber of the multielectrode array simultaneously to detect different analytes on each electrode of the array. A proof of concept ex vivo experiment showed that the multielectrode array could record redox activity in different areas within the mouse caudate putamen and detect dopamine in a 3-mm2 area. To our knowledge, this is the first use of the multielectrode array paired with a commercially available multichannel potentiostat for multi-waveform application and neurotransmitter co-detection. This novel array may aid in future studies to better understand complex brain heterogeneity, the dynamic neurochemical environment, and how disease states or drugs affect separate brain areas concurrently. Graphical abstract.

Simultaneous determination of cellular adenosine nucleotides, malondialdehyde, and uric acid using HPLC.

Adenine nucleotides and malondialdehyde (MDA) are key components involved in energy metabolism and reactive oxygen species (ROS) production. Measuring the levels of these components at the same time would be critical in studying mitochondrial functions. We have established a HPLC method to simultaneously measure adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, MDA, and uric acid (UA). The samples were treated with perchloric acid followed by centrifugation. After neutralization, the supernatant was subjected to HPLC determination. HPLC was performed using a C18 chromatographic column, isocratic elusion, and UV detection. The detection and quantification limits for these components were determined with standard solutions. The precision, repeatability, and 24-h stability were evaluated using cellular samples, and their relative standard deviations were all within 2%. The reproducibility and efficiency were confirmed with sample recovery tests and the observed oxidative effects of H2 O2 on Jurkat cells. With this method, we discovered the dependence of energy and oxidative states on the density of Jurkat cells cultured in suspension. We also found a significant correlation between UA in serum and that in saliva. These results indicate that this method has good accuracy and applicability. It can be used in biological, pharmacological, and clinical studies, especially those involving mitochondria, ROS, and purinergic signaling.

Anomalous Reverse Transcription through Chemical Modifications in Polyadenosine Stretches.

Thermostable reverse transcriptases are workhorse enzymes underlying nearly all modern techniques for RNA structure mapping and for the transcriptome-wide discovery of RNA chemical modifications. Despite their wide use, these enzymes' behaviors at chemical modified nucleotides remain poorly understood. Wellington-Oguri et al. recently reported an apparent loss of chemical modification within putatively unstructured polyadenosine stretches modified by dimethyl sulfate or 2' hydroxyl acylation, as probed by reverse transcription. Here, reanalysis of these and other publicly available data, capillary electrophoresis experiments on chemically modified RNAs, and nuclear magnetic resonance spectroscopy on (A)12 and variants show that this effect is unlikely to arise from an unusual structure of polyadenosine. Instead, tests of different reverse transcriptases on chemically modified RNAs and molecules synthesized with single 1-methyladenosines implicate a previously uncharacterized reverse transcriptase behavior: near-quantitative bypass through chemical modifications within polyadenosine stretches. All tested natural and engineered reverse transcriptases (MMLV; SuperScript II, III, and IV; TGIRT-III; and MarathonRT) exhibit this anomalous bypass behavior. Accurate DMS-guided structure modeling of the polyadenylated HIV-1 3' untranslated region requires taking into account this anomaly. Our results suggest that poly(rA-dT) hybrid duplexes can trigger an unexpectedly effective reverse transcriptase bypass and that chemical modifications in mRNA poly(A) tails may be generally undercounted.

Sbp1 modulates the translation of Pab1 mRNA in a poly(A)- and RGG-dependent manner.

RNA-binding protein Sbp1 facilitates the decapping pathway in mRNA metabolism and inhibits global mRNA translation by an unclear mechanism. Here we report molecular interactions responsible for Sbp1-mediated translation inhibition of mRNA encoding the polyadenosine-binding protein (Pab1), an essential translation factor that stimulates mRNA translation and inhibits mRNA decapping in eukaryotic cells. We demonstrate that the two distal RRMs of Sbp1 bind to the poly(A) sequence in the 5'UTR of the Pab1 mRNA specifically and cooperatively while the central RGG domain of the protein interacts directly with Pab1. Furthermore, methylation of arginines in the RGG domain abolishes the protein-protein interaction and the inhibitory effect of Sbp1 on translation initiation of Pab1 mRNA. Based on these results, the underlying mechanism for Sbp1-specific translational regulation is proposed. The functional differences of Sbp1 and RGG repeats alone on transcript-specific translation were observed, and a comparison of the results suggests the importance of remodeling the 5'UTR by RNA-binding proteins in mRNA translation.