Isolation of Endogenously Assembled RNA-Protein Complexes Using Affinity Purification Based on Streptavidin Aptamer S1

Efficient isolation of endogenously assembled viral RNA-protein complexes is essential for understanding virus replication mechanisms. We have developed an affinity purification strategy based on an RNA affinity tag that allows large-scale preparation of native viral RNA-binding proteins (RBPs). The streptavidin-binding aptamer S1 sequence was inserted into the 3′ end of dengue virus (DENV) 5′–3′ UTR RNA, and the DENV RNA UTR fused to the S1 RNA aptamer was expressed in living mammalian cells. This allowed endogenous viral ribonucleoprotein (RNP) assembly and isolation of RNPs from whole cell extract, through binding the S1 aptamer to streptavidin magnetic beads. Several novel host DENV RBPs were subsequently identified by liquid chromatography with tandem mass spectrometry (LC-MS/MS), including RPS8, which we further implicate in DENV replication. We proposed efficient S1 aptamer-based isolation of viral assembled RNPs from living mammalian cells will be generally applicable to the purification of high- and low-affinity RBPs and RNPs under endogenous conditions.     

Therapeutic Targeting of Alternative RNA Splicing in Gastrointestinal Malignancies and Other Cancers

Recent comprehensive genomic studies including single-cell RNA sequencing and characterization have revealed multiple processes by which protein-coding and noncoding RNA processing are dysregulated in many cancers. More specifically, the abnormal regulation of mRNA and precursor mRNA (pre-mRNA) processing, which includes the removal of introns by splicing, is frequently altered in tumors, producing multiple different isoforms and diversifying protein expression. These alterations in RNA processing result in numerous cancer-specific mRNAs and pathogenically spliced events that generate altered levels of normal proteins or proteins with new functions, leading to the activation of oncogenes or the inactivation of tumor suppressor genes. Abnormally spliced pre-mRNAs are also associated with resistance to cancer treatment, and certain cancers are highly sensitive to the pharmacological inhibition of splicing. The discovery of these alterations in RNA processing has not only provided new insights into cancer pathogenesis but identified novel therapeutic vulnerabilities and therapeutic opportunities in targeting these aberrations in various ways (e.g., small molecules, splice-switching oligonucleotides (SSOs), and protein therapies) to modulate alternative RNA splicing or other RNA processing and modification mechanisms. Some of these strategies are currently progressing toward clinical development or are already in clinical trials. Additionally, tumor-specific neoantigens produced from these pathogenically spliced events and other abnormal RNA processes provide a potentially extensive source of tumor-specific therapeutic antigens (TAs) for targeted cancer immunotherapy. Moreover, a better understanding of the molecular mechanisms associated with aberrant RNA processes and the biological impact they play might provide insights into cancer initiation, progression, and metastasis. Our goal is to highlight key alternative RNA splicing and processing mechanisms and their roles in cancer pathophysiology as well as emerging therapeutic alternative splicing targets in cancer, particularly in gastrointestinal (GI) malignancies.     

The Role of Phytohormones in Plant Response to Flooding

Climatic variations influence the morphological, physiological, biological, and biochemical states of plants. Plant responses to abiotic stress include biochemical adjustments, regulation of proteins, molecular mechanisms, and alteration of post-translational modifications, as well as signal transduction. Among the various abiotic stresses, flooding stress adversely affects the growth of plants, including various economically important crops. Biochemical and biological techniques, including proteomic techniques, provide a thorough understanding of the molecular mechanisms during flooding conditions. In particular, plants can cope with flooding conditions by embracing an orchestrated set of morphological adaptations and physiological adjustments that are regulated by an elaborate hormonal signaling network. With the help of these findings, the main objective is to identify plant responses to flooding and utilize that information for the development of flood-tolerant plants. This review provides an insight into the role of phytohormones in plant response mechanisms to flooding stress, as well as different mitigation strategies that can be successfully administered to improve plant growth during stress exposure. Ultimately, this review will expedite marker-assisted genetic enhancement studies in crops for developing high-yield lines or varieties with flood tolerance.     

Roles of Physicochemical and Structural Properties of RNA-Binding Proteins in Predicting the Activities of Trans-Acting Splicing Factors with Machine Learning

Trans-acting splicing factors play a pivotal role in modulating alternative splicing by specifically binding to cis-elements in pre-mRNAs. There are approximately 1500 RNA-binding proteins (RBPs) in the human genome, but the activities of these RBPs in alternative splicing are unknown. Since determining RBP activities through experimental methods is expensive and time consuming, the development of an efficient computational method for predicting the activities of RBPs in alternative splicing from their sequences is of great practical importance. Recently, a machine learning model for predicting the activities of splicing factors was built based on features of single and dual amino acid compositions. Here, we explored the role of physicochemical and structural properties in predicting their activities in alternative splicing using machine learning approaches and found that the prediction performance is significantly improved by including these properties. By combining the minimum redundancy–maximum relevance (mRMR) method and forward feature searching strategy, a promising feature subset with 24 features was obtained to predict the activities of RBPs. The feature subset consists of 16 dual amino acid compositions, 5 physicochemical features, and 3 structural features. The physicochemical and structural properties were as important as the sequence composition features for an accurate prediction of the activities of splicing factors. The hydrophobicity and distribution of coil are suggested to be the key physicochemical and structural features, respectively.