Molecular Basis and Consequences of the Cytochrome c-tRNA Interaction.

The intrinsic apoptosis pathway occurs through the release of mitochondrial cytochrome c to the cytosol, where it promotes activation of the caspase family of proteases. The observation that tRNA binds to cytochrome c revealed a previously unexpected mode of apoptotic regulation. However, the molecular characteristics of this interaction, and its impact on each interaction partner, are not well understood. Using a novel fluorescence assay, we show here that cytochrome c binds to tRNA with an affinity comparable with other tRNA-protein binding interactions and with a molecular ratio of ∼3:1. Cytochrome c recognizes the tertiary structural features of tRNA, particularly in the core region. This binding is independent of the charging state of tRNA but is regulated by the redox state of cytochrome c. Compared with reduced cytochrome c, oxidized cytochrome c binds to tRNA with a weaker affinity, which correlates with its stronger pro-apoptotic activity. tRNA binding both facilitates cytochrome c reduction and inhibits the peroxidase activity of cytochrome c, which is involved in its release from mitochondria. Together, these findings provide new insights into the cytochrome c-tRNA interaction and apoptotic regulation

Gemin4: A Novel Component of the Smn Complex That Is Found in Both Gems and Nucleoli

The survival of motor neurons (SMN) protein, the product of the neurodegenerative disease spinal muscular atrophy (SMA) gene, is localized both in the cytoplasm and in discrete nuclear bodies called gems. In both compartments SMN is part of a large complex that contains several proteins including Gemin2 (formerly SIP1) and the DEAD box protein Gemin3. In the cytoplasm, the SMN complex is associated with snRNP Sm core proteins and plays a critical role in spliceosomal snRNP assembly. In the nucleus, SMN is required for pre-mRNA splicing by serving in the regeneration of spliceosomes. These functions are likely impaired in cells of SMA patients because they have reduced levels of functional SMN. Here, we report the identification by nanoelectrospray mass spectrometry of a novel component of the SMN complex that we name Gemin4. Gemin4 is associated in vivo with the SMN complex through a direct interaction with Gemin3. The tight interaction of Gemin4 with Gemin3 suggests that it could serve as a cofactor of this DEAD box protein. Gemin4 also interacts directly with several of the Sm core proteins. Monoclonal antibodies against Gemin4 efficiently immunoprecipitate the spliceosomal U snRNAs U1 and U5 from Xenopus oocytes cytoplasm. Immunolocalization experiments show that Gemin4 is colocalized with SMN in the cytoplasm and in gems. Interestingly, Gemin4 is also detected in the nucleoli, suggesting that the SMN complex may also function in preribosomal RNA processing or ribosome assembly

Ars2 Links the Nuclear Cap-Binding Complex to RNA Interference and Cell Proliferation

SummaryHere we identify a component of the nuclear RNA cap-binding complex (CBC), Ars2, that is important for miRNA biogenesis and critical for cell proliferation. Unlike other components of the CBC, Ars2 expression is linked to the proliferative state of the cell. Deletion of Ars2 is developmentally lethal, and deletion in adult mice led to bone marrow failure whereas parenchymal organs composed of nonproliferating cells were unaffected. Depletion of Ars2 or CBP80 from proliferating cells impaired miRNA-mediated repression and led to alterations in primary miRNA processing in the nucleus. Ars2 depletion also reduced the levels of several miRNAs, including miR-21, let-7, and miR-155, that are implicated in cellular transformation. These findings provide evidence for a role for Ars2 in RNA interference regulation during cell proliferation

Diverse functions of RNA -binding proteins in the biogenesis of ribonucleoprotein particles

RNA-binding proteins play crucial roles in the biogenesis of RNPs. In eukaryotes, mature mRNAs are produced by pre-mRNA processing in the cell nucleus. Along this pathway, heterogeneous nuclear ribonucleoprotein (hnRNP) complexes are formed due to association of numerous RNA-binding proteins with these transcripts. Here, I investigated the dynamic change of protein composition in the hnRNP complexes during pre-mRNA processing and demonstrated that pre-mRNA splicing creates novel mRNP complexes distinct from hnRNP complexes. One of the components of mRNP complex, Y14, associates preferentially with spliced mRNAs but not with pre-mRNAs, introns, or mRNAs produced from intronless cDNAs. Y14 binds to both nuclear mRNAs and newly exported cytoplasmic mRNAs. Microinjections of pre-mRNAs into Xenopus oocytes followed by immunoprecipitations of RNase fragmented mRNAs showed that Y14 associates upstream of exon-exon junctions. These findings demonstrate that the splicing-dependent binding of Y14 provides a position-specific molecular memory that communicates to the cytoplasm the location of exon-intron boundaries and further suggest that pre-mRNA splicing serves not only to remove introns but also to imprint a unique mRNP. Splicing of pre-mRNA takes place in the spliceosome, a dynamic assembly of pre-mRNA, small nuclear RNPs (snRNPs), and numerous protein factors. The biogenesis of spliceosomal U snRNPs, the catalytic core of the spliceosome, is a complex process. Important and unexpected insights into this process of snRNP assembly emerged from studies on the function of the SMN complex. Spliceosomal snRNPs have a common core comprised of seven Sm proteins and each snRNA. To mediate the assembly of snRNPs, the SMN complex must have the capacity to bring together both the Sm proteins and the U snRNAs. Here, I describe that the SMN complex interacts directly with the spliceosomal U snRNAs. In U1 snRNA, the stem-loop 1 domain (SL1) is necessary and sufficient for SMN complex binding. Substitution of three nucleotides in the loop of SL1 (SL1A3) abolishes SMN interaction, and the corresponding U1 snRNA is impaired in U1 snRNP biogenesis. In addition, an excess of SL1 but not SL1A3 interferes the binding of the SMN complex with the U snRNAs and therefore inhibits the snRNPs assembly. These findings demonstrate that the interaction of the SMN complex with the U snRNAs is crucial for U snRNPs biogenesis further supporting the direct role of the SMN complex in RNP biogenesis

Diverse functions of RNA -binding proteins in the biogenesis of ribonucleoprotein particles

RNA-binding proteins play crucial roles in the biogenesis of RNPs. In eukaryotes, mature mRNAs are produced by pre-mRNA processing in the cell nucleus. Along this pathway, heterogeneous nuclear ribonucleoprotein (hnRNP) complexes are formed due to association of numerous RNA-binding proteins with these transcripts. Here, I investigated the dynamic change of protein composition in the hnRNP complexes during pre-mRNA processing and demonstrated that pre-mRNA splicing creates novel mRNP complexes distinct from hnRNP complexes. One of the components of mRNP complex, Y14, associates preferentially with spliced mRNAs but not with pre-mRNAs, introns, or mRNAs produced from intronless cDNAs. Y14 binds to both nuclear mRNAs and newly exported cytoplasmic mRNAs. Microinjections of pre-mRNAs into Xenopus oocytes followed by immunoprecipitations of RNase fragmented mRNAs showed that Y14 associates upstream of exon-exon junctions. These findings demonstrate that the splicing-dependent binding of Y14 provides a position-specific molecular memory that communicates to the cytoplasm the location of exon-intron boundaries and further suggest that pre-mRNA splicing serves not only to remove introns but also to imprint a unique mRNP. Splicing of pre-mRNA takes place in the spliceosome, a dynamic assembly of pre-mRNA, small nuclear RNPs (snRNPs), and numerous protein factors. The biogenesis of spliceosomal U snRNPs, the catalytic core of the spliceosome, is a complex process. Important and unexpected insights into this process of snRNP assembly emerged from studies on the function of the SMN complex. Spliceosomal snRNPs have a common core comprised of seven Sm proteins and each snRNA. To mediate the assembly of snRNPs, the SMN complex must have the capacity to bring together both the Sm proteins and the U snRNAs. Here, I describe that the SMN complex interacts directly with the spliceosomal U snRNAs. In U1 snRNA, the stem-loop 1 domain (SL1) is necessary and sufficient for SMN complex binding. Substitution of three nucleotides in the loop of SL1 (SL1A3) abolishes SMN interaction, and the corresponding U1 snRNA is impaired in U1 snRNP biogenesis. In addition, an excess of SL1 but not SL1A3 interferes the binding of the SMN complex with the U snRNAs and therefore inhibits the snRNPs assembly. These findings demonstrate that the interaction of the SMN complex with the U snRNAs is crucial for U snRNPs biogenesis further supporting the direct role of the SMN complex in RNP biogenesis

Development of a Simple and Powerful Analytical Method for Formaldehyde Detection and Quantitation in Blood Samples

Human beings are easily exposed to formaldehyde (FA) in a living environment. Entry of FA into the human body can have adverse effects on human health, depending on the FA concentration. Thus, a quantitative analysis of FA in blood is necessary in order to estimate its effect on the human body. In this study, a simple and rapid analytical method for the quantitation of FA in blood was developed. The total analysis time, including the pretreatment procedure, was less than 20 min. To ensure a stable analysis, blood samples were stabilized using tripotassium ethylenediaminetetraacetic acid solution, and FA was selectively derivatized using 2,4-dinitrophenylhydrazine as pretreatment procedures. The pretreated samples were analyzed using a high-performance liquid chromatography-UV system, which is the most common choice for analyzing small-molecule aldehydes like formaldehyde. Verification of the pretreatment methods (stabilization and derivatization) using FA standards confirmed that the pretreatment methods are highly reliable in the calibration range 0.012–5.761 ng μL–1 (slope = 684,898, R2 = 0.9998, and limit of detection = 0.251 pg·μL–1). Analysis of FA in the blood samples of a Yucatan minipig using the new method revealed an average FA concentration of 1.98 ± 0.34 ng μL–1 (n = 3). Blood samples spiked with FA standards were analyzed, and the FA concentrations were found to be similar to the theoretical concentrations (2.16 ± 0.81% difference). The method reported herein can quantitatively analyze FA in blood at a sub-nanogram level within a short period of time and is validated for application in blood analysis

Sequence-specific interaction of U1 snRNA with the SMN complex

The survival of motor neurons (SMN) protein complex functions in the biogenesis of spliceosomal small nuclear ribonucleoprotein particles (snRNPs) and prob ably other RNPs. All spliceosomal snRNPs have a common core of seven Sm proteins. To mediate the assembly of snRNPs, the SMN complex must be able to bring together Sm proteins with U snRNAs. We showed previously that SMN and other components of the SMN complex interact directly with several Sm proteins. Here, we show that the SMN complex also interacts specifically with U1 snRNA. The stem–loop 1 domain of U1 (SL1) is necessary and sufficient for SMN complex binding in vivo and in vitro. Substitution of three nucleotides in the SL1 loop (SL1A3) abolishes SMN interaction, and the corresponding U1 snRNA (U1A3) is impaired in U1 snRNP biogenesis. Microinjection of excess SL1 but not SL1A3 into Xenopus oocytes inhibits SMN complex binding to U1 snRNA and U1 snRNP assembly. These findings indicate that SMN complex interaction with SL1 is sequence-specific and critical for U1 snRNP biogenesis, further supporting the direct role of the SMN complex in RNP biogenesis

Specific Sequence Features, Recognized by the SMN Complex, Identify snRNAs and Determine Their Fate as snRNPs

The survival of motor neurons (SMN) complex is essential for the biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) as it binds to and delivers Sm proteins for assembly of Sm cores on the abundant small nuclear RNAs (snRNAs). Using the conserved snRNAs encoded by the lymphotropic Herpesvirus saimiri (HVS), we determined the specific sequence and structural features of RNAs for binding to the SMN complex and for Sm core assembly. We show that the minimal SMN complex-binding domain in snRNAs, except U1, is comprised of an Sm site (AUUUUUG) and an adjacent 3′ stem-loop. The adenosine and the first and third uridines of the Sm site are particularly critical for binding of the SMN complex, which directly contacts the backbone phosphates of these uridines. The specific sequence of the adjacent stem (7 to 12 base pairs)-loop (4 to 17 nucleotides) is not important for SMN complex binding, but it must be located within a short distance of the 3′ end of the RNA for an Sm core to assemble. Importantly, these defining characteristics are discerned by the SMN complex and not by the Sm proteins, which can bind to and assemble on an Sm site sequence alone. These findings demonstrate that the SMN complex is the identifier, as well as assembler, of the abundant class of snRNAs in cells because it is able to recognize an snRNP code that they contain

Alternative Polyadenylation in Human Diseases

Varying length of messenger RNA (mRNA) 3′-untranslated region is generated by alternating the usage of polyadenylation sites during pre-mRNA processing. It is prevalent through all eukaryotes and has emerged as a key mechanism for controlling gene expression. Alternative polyadenylation (APA) plays an important role for cell growth, proliferation, and differentiation. In this review, we discuss the functions of APA related with various physiological conditions including cellular metabolism, mRNA processing, and protein diversity in a variety of disease models. We also discuss the molecular mechanisms underlying APA regulation, such as variations in the concentration of mRNA processing factors and RNA-binding proteins, as well as global transcriptome changes under cellular signaling pathway

Network-based machine learning and graph theory algorithms for precision oncology.

Network-based analytics plays an increasingly important role in precision oncology. Growing evidence in recent studies suggests that cancer can be better understood through mutated or dysregulated pathways or networks rather than individual mutations and that the efficacy of repositioned drugs can be inferred from disease modules in molecular networks. This article reviews network-based machine learning and graph theory algorithms for integrative analysis of personal genomic data and biomedical knowledge bases to identify tumor-specific molecular mechanisms, candidate targets and repositioned drugs for personalized treatment. The review focuses on the algorithmic design and mathematical formulation of these methods to facilitate applications and implementations of network-based analysis in the practice of precision oncology. We review the methods applied in three scenarios to integrate genomic data and network models in different analysis pipelines, and we examine three categories of network-based approaches for repositioning drugs in drug-disease-gene networks. In addition, we perform a comprehensive subnetwork/pathway analysis of mutations in 31 cancer genome projects in the Cancer Genome Atlas and present a detailed case study on ovarian cancer. Finally, we discuss interesting observations, potential pitfalls and future directions in network-based precision oncology