Characterization of RNA binding specificity of the Drosophila sex-lethal protein by in vitro ligand selection.

The Drosophila sex-lethal (Sxl) protein, a regulator of somatic sexual differentiation, is an RNA binding protein with two potential RNA recognition motifs (RRMs). It is thought to exert its function on splicing by binding to specific RNA sequences within Sxl and transformer (tra) pre-mRNAs. To examine the Sxl RNA binding specificity in detail, we performed in vitro selection and amplification of ligand RNAs from a random sequence pool on the basis of affinity with Sxl protein. After three cycles of selection and amplification, we cloned and sequenced 17 cDNAs corresponding to the RNAs selected in vitro. Sequencing showed that most of the RNAs selected contain polyuridine stretches surrounded by purine residues. In vitro binding analysis revealed that the sequences of the in vitro selected RNAs with relatively high affinity for Sxl show similarity to that of the Sxl- and tra-regulated acceptor regions, including the invariant AG sequence for splicing. These results suggest that Sxl recognizes and preferentially binds to a polyuridine stretch with a downstream AG sequence.     

Divergent RNA binding specificity of yeast Puf2p

PUF proteins bind mRNAs and regulate their translation, stability, and localization. Each PUF protein binds a selective group of mRNAs, enabling their coordinate control. We focus here on the specificity of Puf2p and Puf1p of Saccharomyces cerevisiae, which copurify with overlapping groups of mRNAs. We applied an RNA-adapted version of the DRIM algorithm to identify putative binding sequences for both proteins. We first identified a novel motif in the 3' UTRs of mRNAs previously shown to associate with Puf2p. This motif consisted of two UAAU tetranucleotides separated by a 3-nt linker sequence, which we refer to as the dual UAAU motif. The dual UAAU motif was necessary for binding to Puf2p, as judged by gel shift, yeast three-hybrid, and coimmunoprecipitation from yeast lysates. The UAAU tetranucleotides are required for optimal binding, while the identity and length of the linker sequences are less critical. Puf1p also binds the dual UAAU sequence, consistent with the prior observation that it associates with similar populations of mRNAs. In contrast, three other canonical yeast PUF proteins fail to bind the Puf2p recognition site. The dual UAAU motif is distinct from previously known PUF protein binding sites, which invariably possess a UGU trinucleotide. This study expands the repertoire of cis elements bound by PUF proteins and suggests new modes by which PUF proteins recognize their mRNA targets.     

Drosophila muscleblind is involved in troponin T alternative splicing and apoptosis

Background

Muscleblind-like proteins (MBNL) have been involved in a developmental switch in the use of defined cassette exons. Such transition fails in the CTG repeat expansion disease myotonic dystrophy due, in part, to sequestration of MBNL proteins by CUG repeat RNA. Four protein isoforms (MblA-D) are coded by the unique Drosophila muscleblind gene.

Methodology/Principal Findings

We used evolutionary, genetic and cell culture approaches to study muscleblind (mbl) function in flies. The evolutionary study showed that the MblC protein isoform was readily conserved from nematods to Drosophila, which suggests that it performs the most ancestral muscleblind functions. Overexpression of MblC in the fly eye precursors led to an externally rough eye morphology. This phenotype was used in a genetic screen to identify five dominant suppressors and 13 dominant enhancers including Drosophila CUG-BP1 homolog aret, exon junction complex components tsunagi and Aly, and pro-apoptotic genes Traf1 and reaper. We further investigated Muscleblind implication in apoptosis and splicing regulation. We found missplicing of troponin T in muscleblind mutant pupae and confirmed Muscleblind ability to regulate mouse fast skeletal muscle Troponin T (TnnT3) minigene splicing in human HEK cells. MblC overexpression in the wing imaginal disc activated apoptosis in a spatially restricted manner. Bioinformatics analysis identified a conserved FKRP motif, weakly resembling a sumoylation target site, in the MblC-specific sequence. Site-directed mutagenesis of the motif revealed no change in activity of mutant MblC on TnnT3 minigene splicing or aberrant binding to CUG repeat RNA, but altered the ability of the protein to form perinuclear aggregates and enhanced cell death-inducing activity of MblC overexpression.

Conclusions/Significance

Taken together our genetic approach identify cellular processes influenced by Muscleblind function, whereas in vivo and cell culture experiments define Drosophila troponin T as a new Muscleblind target, reveal a potential involvement of MblC in programmed cell death and recognize the FKRP motif as a putative regulator of MblC function and/or subcellular location in the cell.     

The role of disorder in RNA binding affinity and specificity

Most RNA-binding modules are small and bind few nucleotides. RNA-binding proteins typically attain the physiological specificity and affinity for their RNA targets by combining several RNA-binding modules. Here, we review how disordered linkers connecting RNA-binding modules govern the specificity and affinity of RNA–protein interactions by regulating the effective concentration of these modules and their relative orientation. RNA-binding proteins also often contain extended intrinsically disordered regions that mediate protein–protein and RNA–protein interactions with multiple partners. We discuss how these regions can connect proteins and RNA resulting in heterogeneous higher-order assemblies such as membrane-less compartments and amyloid-like structures that have the characteristics of multi-modular entities. The assembled state generates additional RNA-binding specificity and affinity properties that contribute to further the function of RNA-binding proteins within the cellular environment.     

Insights into the RNA quadruplex binding specificity of DDX21

Guanine quadruplexes can form in both DNA and RNA and influence many biological processes through various protein interactions. The DEAD-box RNA helicase protein DDX21 has been shown to bind and remodel RNA quadruplexes but little is known about its specificity for different quadruplex species. Previous reports have suggested DDX21 may interact with telomeric repeat containing RNA quadruplex (TERRA), an integral component of the telomere that contributes to telomeric heterochromatin formation and telomere length regulation. Here we report that the C-terminus of DDX21 directly interacts with TERRA. We use, for the first time, 2D saturation transfer difference NMR to map the protein binding site on a ribonucleic acid species and show that the quadruplex binding domain of DDX21 interacts primarily with the phosphoribose backbone of quadruplexes. Furthermore, by mutating the 2′OH of loop nucleotides we can drastically reduce DDX21's affinity for quadruplex, indicating that the recognition of quadruplex and specificity for TERRA is mediated by interactions with the 2′OH of loop nucleotides.     

Cooperativity in RNA-protein interactions: global analysis of RNA binding specificity

The control and function of RNA are governed by the specificity of RNA binding proteins. Here, we describe a method for global unbiased analysis of RNA-protein interactions that uses in vitro selection, high-throughput sequencing, and sequence-specificity landscapes. The method yields affinities for a vast array of RNAs in a single experiment, including both low- and high-affinity sites. It is reproducible and accurate. Using this approach,we analyzed members of the PUF (Pumilio and FBF) family of eukaryotic mRNA regulators. Our data identify effects of a specific protein partner on PUF-RNA interactions, reveal subsets of target sites not previously detected, and demonstrate that designer PUF proteins can precisely alter specificity. The approach described here is, in principle, broadly applicable for analysis of any molecule that binds RNA, including proteins, nucleic acids, and small molecules.     

Identification of RNA binding sequences of Drosophila SR protein DX16

Dxl6 is a member of the Drosophila melanogaster SR protein family, a group of nuclear proteins that are both essential splicing factors and specific splicing regulators. To get more insight of Dx16 function, we generated the monoclonal antibody against Dx16 and determined its expression pattern and subcellular location. It is mainly expressed in the nucleus of CNS in Drosophila embryos. In order to investigate the RNA-binding specificity of Dxl6, Dxl6-binding RNAs were identified by SELEX screen by using recombinant Dxl6 N-terminus protein as the target. These RNAs contained a consensus motif. Some pre-mRNAs from the corresponding genes showed splicing defects in the Dxl6-P-element insertional mutant fly. These results indicate that Dxl6 has unique functions in the removal of some introns during development.     

RNA binding assays for Tat-derived peptides: implications for specificity.

RNA recognition by the HIV Tat protein is mediated in part by an arginine- and lysine-rich basic subdomain implicated as a signature element in proteins that bind RNA. Relative RNA binding affinities for a 14-residue peptide derived from Tat that spans the basic region are determined using a competition protocol. Binding specificity is compared with complexation by a 38-residue model for the RNA binding domain of Tat using the same approach. Binding strength for the minimal (14 residue) peptide is correlated with that for the longer peptide: both peptides recognize a short, bulged duplex. However, the shorter peptide dissociates more rapidly from the wild-type site and discriminates less well between nonspecific (double-stranded RNA) and specific sites. Relative dissociation constants for 38-residue peptide determined from direct partition and competition assays differ; the former assay consistently predicts stronger discrimination against RNAs with mutations in the stems flanking the bulge. Differences between the two assays are reconciled in terms of contributions from labile binding which is unstable to native gel electrophoresis. Kinetic stability may constitute a major specificity determinant for basic subdomain-mediated recognition of RNA.     

A Caenorhabditis elegans PUF protein family with distinct RNA binding specificity

PUF proteins comprise a highly conserved family of sequence-specific RNA binding proteins that regulate target mRNAs via binding directly to their 3'UTRs. The Caenorhabditis elegans genome encodes several PUF proteins, which cluster into four groups based on sequence similarity; all share amino acids that interact with the RNA in the cocrystal of human Pumilio with RNA. Members of the FBF and the PUF-8/9 groups bind different but related RNA sequences. We focus here on the binding specificity of representatives of a third cluster, comprising PUF-5, -6, and -7. We performed in vivo selection experiments using the yeast three-hybrid system to identify RNA sequences that bind PUF-5 and PUF-6, and we confirmed binding to optimal sites in vitro. The consensus sequences derived from the screens are similar for PUF-5 and PUF-6 but differ from those of the FBF or PUF-8/-9 groups. Similarly, neither PUF-5 nor PUF-6 bind the recognition sites preferred by the other clusters. Mutagenesis studies confirmed the unique RNA specificity of PUF-5/-6. Using the PUF-5 consensus derived from our experiments, we searched a database of C. elegans 3'UTRs to identify potential targets of PUF-5, several of which indeed bind PUF-5. Therefore the consensus has predictive value and provides a route to finding genuine targets of these proteins.     

Identification of an RNA binding specificity for the potential splicing factor TLS

The TLS/FUS gene is involved in a recurrent chromosomal translocation in human myxoid liposarcomas. We previously reported that TLS is a potential splicing regulator able to modulate the 5'-splice site selection in an E1A pre-mRNA. Using an in vitro selection procedure, we investigated whether TLS exhibits a specificity with regard to RNA recognition. The RNAs selected by TLS share a common GGUG motif. Mutation of a G or U residue within this motif abolishes the interaction of TLS with the selected RNAs. We showed that TLS can bind GGUG-containing RNAs with a 250 nm affinity. By UV cross-linking/competition and immunoprecipitation experiments, we demonstrated that TLS recognizes a GGUG-containing RNA in nuclear extracts. Each one of the RNA binding domains (the three RGG boxes and the RNA recognition motif) contributes to the specificity of the TLS.RNA interaction, whereas only RRM and RGG2-3 participate to the E1A alternative splicing in vivo. The specificity of the TLS.RNA interaction was also observed using as natural pre-mRNA, the G-rich IVSB7 intron of the beta-tropomyosin pre-mRNA. Moreover, we determined that RNA binding specificities of TLS and high nuclear ribonucleoprotein A1 were different. Hence, our results help define the role of the specific interaction of TLS with RNA during the splicing process of a pre-mRNA.     

Two different combinations of RNA-binding domains determine the RNA binding specificity of nucleolin

Nucleolin is an abundant nucleolar protein involved in several steps of ribosome biogenesis. The protein is highly conserved through evolution and possesses four RNA-binding domains (RBD), which are likely to determine its RNA binding specificity. Previous studies have shown that nucleolin interacts with two different RNA targets. The first is a small stem-loop structure, the nucleolin recognition element (NRE), found all along the pre-ribosomal RNA. The second is a short single-stranded RNA sequence, the evolutionary conserved motif (ECM), located five nucleotides downstream of the first processing site in the pre-ribosomal RNA 5' external transcribed spacer. Biochemical, genetic, and structural studies have shown that the first two RBD of nucleolin are necessary and sufficient for the specific interaction of nucleolin with the NRE motif. In this work, we have studied the interaction of nucleolin with the ECM sequence. Deletion and mutational analyses showed that all four RBDs of hamster nucleolin were required for the interaction with the ECM sequence. This RNA binding specificity is conserved between hamster and Xenopus laevis, whereas the Xenopus protein does not interact with the NRE. Nucleolin is the first example of a protein that requires four RBDs for its interaction with an RNA target, demonstrating that a single protein can use different combinations of RBD to interact specifically with several RNA sequences.     

RNA binding specificity of Unr, a protein with five cold shock domains

The human unr gene encodes an 85 kDa protein which contains five cold shock domains (CSD). The capacity of Unr to interact in vitro with RNA and its intracellular localization suggest that Unr could be involved in some aspect of cytoplasmic mRNA metabolism. As a step towards identification of Unr mRNA targets, we investigated the RNA-binding specificity of Unr by an in vitro selection approach (SELEX). Purine-rich sequences were selected by Unr, leading to the identification of two related consensus sequences characterized by a conserved core motif AAGUA/G or AACG downstream of a purine stretch. These consensus sequences are 11-14 nt long and appear unstructured. RNAs containing a consensus sequence were bound specifically by Unr with an apparent dissociation constant of 1 x 10(-8) M and both elements, the 5' purine stretch and the core motif, were shown to contribute to the high affinity. When the N-terminal and C-terminal CSD were analyzed individually, they exhibited a lower affinity than Unr for winner sequences (5- and 100-fold, respectively) but with similar binding specificity. Two combinations of CSDs, CSD1-2-3 and CSD1*2-3-4-5 were sufficient to achieve the high affinity of Unr, indicating some redundancy between the CSDs of Unr for RNA recognition. The SELEX-generated consensus motifs for Unr differ from the AACAUC motif selected by the Xenopus Y-box factor FRGY2, indicating that a diversity of RNA sequences could be recognized by CSD-containing proteins.     

RNA binding specificity of a Drosophila snRNP protein that shares sequence homology with mammalian U1-A and U2-B" proteins.

We have characterized a recombinant Drosophila melanogaster RNA binding protein, D25, by virtue of its antigenic relationship to mammalian U1 and U2 small nuclear ribonucleoprotein (U snRNP) proteins. Sequence analysis revealed that D25 bears strong similarity to both the human U1 snRNP-A (U1-A) and U2 snRNP-B" (U2-B") proteins. However, at residues known to be critical for the RNA binding specificities of U1-A and U2-B" D25 sequence is more similar to U2-B". Using direct RNA binding assays D25 selected U1 RNA from either HeLa or Drosophila Kc cell total RNA. Furthermore, D25 bound U1 RNA when transfected into mammalian cells. Thus, D25 appears to be a Drosophila homolog of the mammalian U1-A protein, despite its sequence similarity to U2-B".     

Design of molecular beacons for AmpliDet RNA assay--characterization of binding stability and probe specificity

AmpliDet RNA is a real-time diagnostic method, the specificity of which is defined mainly by the molecular beacon (MB). MBs can be characterized according to the stability of their stem-and-loop structures and that of the probe-target duplex via the free energies accompanying their formation. By the application of thermodynamic models, we propose a prediction method for these deltaG(0) parameters, which was compared to experimental analysis. The average absolute discrepancies for deltaG(0)(41) and for the melting temperatures of MB secondary structures were 0.30 +/- 0.26 kcal/mol and 2.15 +/- 1.5 degrees C, respectively. deltaG(0)(41) of probe-target interaction was predicted with a discrepancy of 1.2 +/- 1.0 kcal/mol. To characterize specificity, we formulated a model system with several MBs of highly similar sequence, but different lengths, and template RNAs carrying different types of mutations. We demonstrated the ability to detect a point mutation, or to tolerate one, irrespective of mismatch type. Of the nucleotide analogues tested, universal pyrimidine was found to increase MB tolerance substantially toward polymorphism. In the present study MBs were characterized under AmpliDet RNA conditions, with respect to probe stability, binding strength, and specificity, which led us to propose a design method, useful for probe design for AmpliDet RNA and adaptable to microarrays.