[Show abstract][Hide abstract] ABSTRACT: Long noncoding RNAs (lncRNAs) interact with protein factors to regulate different layers of gene expression transcriptionally or posttranscriptionally. Here we report on the functional consequences of the unanticipated interaction of the RNA binding protein K homology-type splicing regulatory protein (KSRP) with the H19 lncRNA (H19). KSRP directly binds to H19 in the cytoplasm of undifferentiated multipotent mesenchymal C2C12 cells, and this interaction favors KSRP-mediated destabilization of labile transcripts such as myogenin. AKT activation induces KSRP dismissal from H19 and, as a consequence, myogenin mRNA is stabilized while KSRP is repurposed to promote maturation of myogenic microRNAs, thus favoring myogenic differentiation. Our data indicate that H19 operates as a molecular scaffold that facilitates effective association of KSRP with myogenin and other labile transcripts, and we propose that H19 works with KSRP to optimize an AKT-regulated posttranscriptional switch that controls myogenic differentiation.
Proceedings of the National Academy of Sciences 11/2014; 111(47). DOI:10.1073/pnas.1415098111 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: RNA granules have been observed in different organisms, cell types and under different conditions, and their formation is crucial for the mRNA life cycle. However, very little is known about the molecular mechanisms governing their assembly and disassembly. The aggregation-prone LSCRs (low-sequence-complexity regions), and in particular, the polyQ/N-rich regions, have been extensively studied under pathological conditions due to their role in neurodegenerative diseases. In the present review, we discuss recent in vitro, in vivo and computational data that, globally, suggest a role for polyQ/N regions in RNA granule assembly.
Biochemical Society Transactions 08/2014; 42(4):1246-50. DOI:10.1042/BST20140099 · 3.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The single-strand-RNA binding protein KSRP is able to negatively regulate gene expression operating with at least two distinct and integrated postranscriptional mechanisms: i) by promoting decay of unstable mRNAs and ii) by favoring maturation from precursors of select microRNAs (miRNAs) including the prototypical tumor suppressor let-7. Studies performed in primary and cultured cells as well as in mice proved that the ability of KSRP to integrate different levels of gene expression is required for proper immune response, lipid metabolism, cell-fate decisions, tissue regeneration, and DNA damage response.
Seminars in Cell and Developmental Biology 05/2014; 34. DOI:10.1016/j.semcdb.2014.05.004 · 6.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Regulating the expression of individual miRNAs (microRNAs) is important for cell development and function. The up- or down-regulation of the processing of specific miRNA precursors to the mature active form represents one tool to control miRNA concentration and is mediated by proteins that recognize the terminal loop of the RNA precursors. Terminal loop recognition is achieved by the combined action of several RNA-binding domains. The proteins can then regulate the processing by recruiting RNA enzymes, changing the RNA structure and preventing or enhancing the accessibility and processing activity of the core processing complexes. The present review focuses on how terminal loop-binding proteins recognize their RNA targets and mediate their regulatory function(s), and highlights how terminal loop-mediated regulation relates to the broader regulation of mRNA metabolism.
Biochemical Society Transactions 08/2013; 41(4):861-5. DOI:10.1042/BST20130058 · 3.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The discovery of effective new antimalarial agents is urgently needed. One of the most frequently studied molecules anchored
to the parasite surface is the merozoite surface protein-1 (MSP1). At red blood cell invasion MSP1 is proteolytically processed,
and the 19-kDa C-terminal fragment (MSP119) remains on the surface and is taken into the red blood cell, where it is transferred to the food vacuole and persists until
the end of the intracellular cycle. Because a number of specific antibodies inhibit erythrocyte invasion and parasite growth,
MSP119 is therefore a promising target against malaria. Given the structural homology of cupredoxins with the Fab domain of monoclonal
antibodies, an approach combining NMR and isothermal titration calorimetry (ITC) measurements with docking calculations based
on BiGGER is employed on MSP119-cupredoxin complexes. Among the cupredoxins tested, rusticyanin forms a well defined complex with MSP119 at a site that overlaps with the surface recognized by the inhibitory antibodies. The addition of holo-rusticyanin to infected
cells results in parasitemia inhibition, but negligible effects on parasite growth can be observed for apo-rusticyanin and
other proteins of the cupredoxin family. These findings point to rusticyanin as an excellent therapeutic tool for malaria
treatment and provide valuable information for drug design.
[Show abstract][Hide abstract] ABSTRACT: KSRP is a single strand nucleic acid binding protein that controls gene expression at multiple levels. In this review we focus on the recent molecular, cellular, and structural insights into the mRNA decay promoting function of KSRP. We discuss also some aspects of KSRP-dependent microRNA maturation from precursors that are related to its mRNA destabilizing function.
[Show abstract][Hide abstract] ABSTRACT: Let-7 is an important tumor-suppressive microRNA (miRNA) that acts as an on-off switch for cellular differentiation and regulates the expression of a set of human oncogenes. Binding of the human KSRP protein to let-7 miRNA precursors positively regulates their processing to mature let-7, thereby contributing to control of cell proliferation, apoptosis and differentiation. Here we analyze the molecular basis for KSRP-let-7 precursor selectivity and show how the third KH domain of the protein recognizes a G-rich sequence in the pre-let-7 terminal loop and dominates the interaction. The structure of the KH3-RNA complex explains the protein recognition of this noncanonical KH target sequence, and we demonstrate that the specificity of this binding is crucial for the functional interaction between the protein and the miRNA precursor.
[Show abstract][Hide abstract] ABSTRACT: In eukaryotes, RNA-binding proteins that contain multiple K homology (KH) domains play a key role in coordinating the different steps of RNA synthesis, metabolism and localization. Understanding how the different KH modules participate in the recognition of the RNA targets is necessary to dissect the way these proteins operate. We have designed a KH mutant with impaired RNA-binding capability for general use in exploring the role of individual KH domains in the combinatorial functional recognition of RNA targets. A double mutation in the hallmark GxxG loop (GxxG-to-GDDG) impairs nucleic acid binding without compromising the stability of the domain. We analysed the impact of the GDDG mutations in individual KH domains on the functional properties of KSRP as a prototype of multiple KH domain-containing proteins. We show how the GDDG mutant can be used to directly link biophysical information on the sequence specificity of the different KH domains of KSRP and their role in mRNA recognition and decay. This work defines a general molecular biology tool for the investigation of the function of individual KH domains in nucleic acid binding proteins.
Nucleic Acids Research 04/2012; 40(14):6873-86. DOI:10.1093/nar/gks368 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This chapter is dedicated to the investigation of protein–RNA interactions by NMR spectroscopy. In particular, it focuses on the characterization of reversible protein–RNA complexes that regulate mRNA metabolism, where NMR plays a unique role. We describe a set of solution NMR methods used to define protein–RNA interfaces, from the ubiquitous chemical shift perturbation (CSP) to paramagnetic relaxation enhancement (PRE) to the use of intermolecular protein–RNA cross-saturation. We also describe methods to explore affinity and specificity in protein–RNA interactions, as well as to study simultaneous interactions with multiple partners and to define stoichiometry of binding. Our aim is to provide the reader with a streamlined toolkit of NMR-based methods to characterize a protein–RNA complex of interest using standard NMR equipment.
NMR of Biomolecules, 03/2012: pages 218-236; , ISBN: 9783527328505
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNAs) interact with 3'-untranslated regions of messenger RNAs to restrict expression of most protein-coding genes during normal development and cancer. RNA-binding proteins (RBPs) can control the biogenesis, stability and activity of miRNAs. Here we identify RBM38 in a genetic screen for RBPs whose expression controls miRNA access to target mRNAs. RBM38 is induced by p53 and its ability to modulate miRNA-mediated repression is required for proper p53 function. In contrast, RBM38 shows lower propensity to block the action of the p53-controlled miR-34a on SIRT1. Target selectivity is determined by the interaction of RBM38 with uridine-rich regions near miRNA target sequences. Furthermore, in large cohorts of human breast cancer, reduced RBM38 expression by promoter hypermethylation correlates with wild-type p53 status. Thus, our results indicate a novel layer of p53 gene regulation, which is required for its tumour suppressive function.
[Show abstract][Hide abstract] ABSTRACT: microrNNA (mirNAs) are small noncoding RNAs that down-regulate gene expression by reducing stability and/or translation of
target mRNAs. In animals, miRNAs arise from sequential processing of hairpin primary transcripts by two rNase III domain-containing
enzymes, namely Drosha and Dicer, to generate a mature form of about 22 nucleotides. In this chapter we discuss our latest
fndings indicating that KSRP is an integral component of both Drosha and Dicer complexes. KSRP binds to the terminal loop
sequence of a subset of miRNA precursors promoting their maturation. our data indicate that the terminal loop is a pivotal
structure where activators of miRNA processing as well as repressors of miRNA processing act in a coordinated way to convert
cellular signals into changes in miRNA expression processing. This uncovers a new level of complexity of miRNA mechanisms
for gene expression regulation.
[Show abstract][Hide abstract] ABSTRACT: Here we review the role played by transient interactions between multi-functional proteins and their RNA targets in the regulation of mRNA metabolism, and we describe the important function of NMR spectroscopy in the study of these systems. We place emphasis on a general approach for the study of different features of modular multi-domain recognition that uses well-established NMR techniques and that has provided important advances in the general understanding of post-transcriptional regulation.
Biophysics of Structure and Mechanism 04/2011; 40(12):1317-25. DOI:10.1007/s00249-011-0698-z · 2.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: KSRP is a single-strand nucleic acids binding protein that affects RNA fate at multiple levels. KSRP modular structure and its complex pattern of post-translational modifications underpin the interaction with a wide spectrum of RNA target sequences, as well as with other RNA-binding proteins and molecular adaptors. These interactions are important to the regulation of different steps of mRNA metabolism and, in turn, modulate several aspects of cellular proliferation and differentiation. In this review we will discuss in detail KSRP ability to i) promote decay of labile mRNAs interacting with some components of the mRNA decay machinery and ii) favor the maturation of a select group of microRNA precursors.
Frontiers in Bioscience 01/2011; 16(5):1787-96. DOI:10.2741/3821 · 3.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The far upstream element (FUSE) regulatory system promotes a peak in the concentration of c-Myc during cell cycle. First, the FBP transcriptional activator binds to the FUSE DNA element upstream of the c-myc promoter. Then, FBP recruits its specific repressor (FIR), which acts as an on/off transcriptional switch. Here we describe the molecular basis of FIR recruitment, showing that the tandem RNA recognition motifs of FIR provide a platform for independent FUSE DNA and FBP protein binding and explaining the structural basis of the reversibility of the FBP-FIR interaction. We also show that the physical coupling between FBP and FIR is modulated by a flexible linker positioned sequentially to the recruiting element. Our data explain how the FUSE system precisely regulates c-myc transcription and suggest that a small change in FBP-FIR affinity leads to a substantial effect on c-Myc concentration.
[Show abstract][Hide abstract] ABSTRACT: The germline originates from primordial embryonic germ cells which give rise to sperm and egg cells and consequently, to the next generation. Germ cells of many organisms contain electron-dense granules that comprise RNA and proteins indispensable for germline development. Here we review recent reports that provide important insights into the structure and function of crucial RNA and protein components of the granules, including DEAD-box helicases, Tudor domain proteins, Piwi/Argonaute proteins and piRNA. Collectively, these components function in translational control, remodeling of ribonucleoprotein complexes and transposon silencing. Furthermore, they interact with each other by means of conserved structural modules and post-translationally modified amino acids. These data suggest a widespread use of several protein motifs in germline development and further our understanding of other ribonucleoprotein structures, for example, processing bodies and neuronal granules.
[Show abstract][Hide abstract] ABSTRACT: Rna15 is a core subunit of cleavage factor IA (CFIA), an essential transcriptional 3'-end processing factor from Saccharomyces cerevisiae. CFIA is required for polyA site selection/cleavage targeting RNA sequences that surround polyadenylation sites in the 3'-UTR of RNA polymerase-II transcripts. RNA recognition by CFIA is mediated by an RNA recognition motif (RRM) contained in the Rna15 subunit of the complex. We show here that Rna15 has a strong and unexpected preference for GU containing RNAs and reveal the molecular basis for a base selectivity mechanism that accommodates G or U but discriminates against C and A bases. This mode of base selectivity is rather different to that observed in other RRM-RNA structures and is structurally conserved in CstF64, the mammalian counterpart of Rna15. Our observations provide evidence for a highly conserved mechanism of base recognition amongst the 3'-end processing complexes that interact with the U-rich or U/G-rich elements at 3'-end cleavage/polyadenylation sites.
Nucleic Acids Research 05/2010; 38(9):3119-32. DOI:10.1093/nar/gkq002 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: KSRP is a multi-domain RNA-binding protein that recruits the exosome-containing mRNA degradation complex to mRNAs coding for cellular proliferation and inflammatory response factors. The selectivity of this mRNA degradation mechanism relies on KSRP recognition of AU-rich elements in the mRNA 3'UTR, that is mediated by KSRP's KH domains. Our structural analysis shows that the inter-domain linker orients the two central KH domains of KSRP-and their RNA-binding surfaces-creating a two-domain unit. We also show that this inter-domain arrangement is important to the interaction with KSRP's RNA targets.
Nucleic Acids Research 04/2010; 38(15):5193-205. DOI:10.1093/nar/gkq216 · 9.11 Impact Factor