Cancer-Associated Substitutions in RNA Recognition Motifs of PUF60 and U2AF65 Reveal Residues Required for Correct Folding and 3' Splice-Site Selection.

U2AF65 (U2AF2) and PUF60 (PUF60) are splicing factors important for recruitment of the U2 small nuclear ribonucleoprotein to lariat branch points and selection of 3' splice sites (3'ss). Both proteins preferentially bind uridine-rich sequences upstream of 3'ss via their RNA recognition motifs (RRMs). Here, we examined 36 RRM substitutions reported in cancer patients to identify variants that alter 3'ss selection, RNA binding and protein properties. Employing PUF60- and U2AF65-dependent 3'ss previously identified by RNA-seq of depleted cells, we found that 43% (10/23) and 15% (2/13) of independent RRM mutations in U2AF65 and PUF60, respectively, conferred splicing defects. At least three RRM mutations increased skipping of internal U2AF2 (~9%, 2/23) or PUF60 (~8%, 1/13) exons, indicating that cancer-associated RRM mutations can have both cis- and trans-acting effects on splicing. We also report residues required for correct folding/stability of each protein and map functional RRM substitutions on to existing high-resolution structures of U2AF65 and PUF60. These results identify new RRM residues critical for 3'ss selection and provide relatively simple tools to detect clonal RRM mutations that enhance the mRNA isoform diversity.

Staufen1 reads out structure and sequence features in ARF1 dsRNA for target recognition.

Staufen1 (STAU1) is a dsRNA binding protein mediating mRNA transport and localization, translational control and STAU1-mediated mRNA decay (SMD). The STAU1 binding site (SBS) within human ADP-ribosylation factor1 (ARF1) 3'UTR binds STAU1 and this downregulates ARF1 cytoplasmic mRNA levels by SMD. However, how STAU1 recognizes specific mRNA targets is still under debate. Our structure of the ARF1 SBS-STAU1 complex uncovers target recognition by STAU1. STAU1 dsRNA binding domain (dsRBD) 4 interacts with two pyrimidines and one purine from the minor groove side via helix α1, the ß1-ß2 loop anchors the dsRBD at the end of the dsRNA and lysines in helix α2 bind to the phosphodiester backbone from the major groove side. STAU1 dsRBD3 displays the same binding mode with specific recognition of one guanine base. Mutants disrupting minor groove recognition of ARF1 SBS affect in vitro binding and reduce SMD in vivo. Our data thus reveal how STAU1 recognizes minor groove features in dsRNA relevant for target selection.

The Solution Structure of FUS Bound to RNA Reveals a Bipartite Mode of RNA Recognition with Both Sequence and Shape Specificity.

Fused in sarcoma (FUS) is an RNA binding protein involved in regulating many aspects of RNA processing and linked to several neurodegenerative diseases. Transcriptomics studies indicate that FUS binds a large variety of RNA motifs, suggesting that FUS RNA binding might be quite complex. Here, we present solution structures of FUS zinc finger (ZnF) and RNA recognition motif (RRM) domains bound to RNA. These structures show a bipartite binding mode of FUS comprising of sequence-specific recognition of a NGGU motif via the ZnF and an unusual shape recognition of a stem-loop RNA via the RRM. In addition, sequence-independent interactions via the RGG repeats significantly increase binding affinity and promote destabilization of structured RNA conformation, enabling additional binding. We further show that disruption of the RRM and ZnF domains abolishes FUS function in splicing. Altogether, our results rationalize why deciphering the RNA binding mode of FUS has been so challenging.

NMR solution structure determination of large RNA-protein complexes.

Structure determination of RNA-protein complexes is essential for our understanding of the multiple layers of RNA-mediated posttranscriptional regulation of gene expression. Over the past 20years, NMR spectroscopy became a key tool for structural studies of RNA-protein interactions. Here, we review the progress being made in NMR structure determination of large ribonucleoprotein assemblies. We discuss approaches for the design of RNA-protein complexes for NMR structural studies, established and emerging isotope and segmental labeling schemes suitable for large RNPs and how to gain distance restraints from NOEs, PREs and EPR and orientational information from RDCs and SAXS/SANS in such systems. The new combination of NMR measurements with MD simulations and its potential will also be discussed. Application and combination of these various methods for structure determination of large RNPs will be illustrated with three large RNA-protein complexes (>40kDa) and other interesting complexes determined in the past six and a half years.

Efficient large-scale preparation and purification of short single-stranded RNA oligonucleotides.

Sequence-specific RNA recognition by RNA-binding proteins plays a crucial role in the post-translational regulation of gene expression. Biophysical and biochemical studies help to unravel the principles of sequence-specific RNA recognition, but the methods used require large amounts of single-stranded RNA (ssRNA). Here we present a fast and robust method for large-scale preparation and purification of short ssRNA oligonucleotides for biochemical, biophysical, and structural studies. We designed an efficiently folding, self-cleaving hammerhead (HH) ribozyme to prepare ssRNA oligonucleotides. Hammerhead ribozyme RNAs self-cleave with over 95% efficiency during in vitro transcription as a function of magnesium concentration to produce high yields of the desired ssRNA products. The resulting ssRNAs can be purified from crude transcription reactions by denaturing anion-exchange chromatography and then desalted by weak anion-exchange chromatography using volatile ammonium bicarbonate buffer solutions. The ssRNA oligonucleotides produced this way are homogenous, as judged by mass spectrometry (MS), and are suitable for biochemical and biophysical studies. Moreover, for high-resolution NMR structure determination of RNA-protein complexes, our protocol enables efficient preparation of ssRNA oligonucleotides with various isotope-labeling schemes which are not commercially available.

Physicochemical analysis of rotavirus segment 11 supports a 'modified panhandle' structure and not the predicted alternative tRNA-like structure (TRLS).

Rotaviruses are a major cause of acute gastroenteritis, which is often fatal in infants. The viral genome consists of 11 double-stranded RNA segments, but little is known about their cis-acting sequences and structural elements. Covariation studies and phylogenetic analysis exploring the potential structure of RNA11 of rotaviruses suggested that, besides the previously predicted "modified panhandle" structure, the 5' and 3' termini of one of the isoforms of the bovine rotavirus UKtc strain may interact to form a tRNA-like structure (TRLS). Such TRLSs have been identified in RNAs of plant viruses, where they are important for enhancing replication and packaging. However, using tRNA mimicry assays (in vitro aminoacylation and 3'- adenylation), we found no biochemical evidence for tRNA-like functions of RNA11. Capping, synthetic 3' adenylation and manipulation of divalent cation concentrations did not change this finding. NMR studies on a 5'- and 3'-deletion construct of RNA11 containing the putative intra-strand complementary sequences supported a predominant panhandle structure and did not conform to a cloverleaf fold despite the strong evidence for a predicted structure in this conserved region of the viral RNA. Additional viral or cellular factors may be needed to stabilise it into a form with tRNA-like properties.

Molecular basis of UG-rich RNA recognition by the human splicing factor TDP-43.

TDP-43 encodes an alternative-splicing regulator with tandem RNA-recognition motifs (RRMs). The protein regulates cystic fibrosis transmembrane regulator (CFTR) exon 9 splicing through binding to long UG-rich RNA sequences and is found in cytoplasmic inclusions of several neurodegenerative diseases. We solved the solution structure of the TDP-43 RRMs in complex with UG-rich RNA. Ten nucleotides are bound by both RRMs, and six are recognized sequence specifically. Among these, a central G interacts with both RRMs and stabilizes a new tandem RRM arrangement. Mutations that eliminate recognition of this key nucleotide or crucial inter-RRM interactions disrupt RNA binding and TDP-43-dependent splicing regulation. In contrast, point mutations that affect base-specific recognition in either RRM have weaker effects. Our findings reveal not only how TDP-43 recognizes UG repeats but also how RNA binding-dependent inter-RRM interactions are crucial for TDP-43 function.

Bicaudal-D uses a parallel, homodimeric coiled coil with heterotypic registry to coordinate recruitment of cargos to dynein.

Cytoplasmic dynein is the major minus end-directed microtubule motor in eukaryotes. However, there is little structural insight into how different cargos are recognized and linked to the motor complex. Here we describe the 2.2 Å resolution crystal structure of a cargo-binding region of the dynein adaptor Bicaudal-D (BicD), which reveals a parallel coiled-coil homodimer. We identify a shared binding site for two cargo-associated proteins-Rab6 and the RNA-binding protein Egalitarian (Egl)-within a region of the BicD structure with classical, homotypic core packing. Structure-based mutagenesis in Drosophila provides evidence that occupancy of this site drives association of BicD with dynein, thereby coupling motor recruitment to cargo availability. The structure also contains a region in which, remarkably, the same residues in the polypeptide sequence have different heptad registry in each chain. In vitro and in vivo analysis of a classical Drosophila dominant mutation reveals that this heterotypic region regulates the recruitment of dynein to BicD. Our results support a model in which the heterotypic segment is part of a molecular switch that promotes release of BicD autoinhibition following cargo binding to the neighboring, homotypic coiled-coil region. Overall, our data reveal a pivotal role of a highly asymmetric coiled-coil domain in coordinating the assembly of cargo-motor complexes.

Isotope labeling and segmental labeling of larger RNAs for NMR structural studies.

NMR spectroscopy has become substantial in the elucidation of RNA structures and their complexes with other nucleic acids, proteins or small molecules. Almost half of the RNA structures deposited in the Protein Data Bank were determined by NMR spectroscopy, whereas NMR accounts for only 11% for proteins. Recent improvements in isotope labeling of RNA have strongly contributed to the high impact of NMR in RNA structure determination. In this book chapter, we review the advances in isotope labeling of RNA focusing on larger RNAs. We start by discussing several ways for the production and purification of large quantities of pure isotope labeled RNA. We continue by reviewing different strategies for selective deuteration of nucleotides. Finally, we present a comparison of several approaches for segmental isotope labeling of RNA. Selective deuteration of nucleotides in combination with segmental isotope labeling is paving the path for studying RNAs of ever increasing size.

Structural analysis of an eIF3 subcomplex reveals conserved interactions required for a stable and proper translation pre-initiation complex assembly.

Translation initiation factor eIF3 acts as the key orchestrator of the canonical initiation pathway in eukaryotes, yet its structure is greatly unexplored. We report the 2.2 Å resolution crystal structure of the complex between the yeast seven-bladed ß-propeller eIF3i/TIF34 and a C-terminal α-helix of eIF3b/PRT1, which reveals universally conserved interactions. Mutating these interactions displays severe growth defects and eliminates association of eIF3i/TIF34 and strikingly also eIF3g/TIF35 with eIF3 and 40S subunits in vivo. Unexpectedly, 40S-association of the remaining eIF3 subcomplex and eIF5 is likewise destabilized resulting in formation of aberrant pre-initiation complexes (PICs) containing eIF2 and eIF1, which critically compromises scanning arrest on mRNA at its AUG start codon suggesting that the contacts between mRNA and ribosomal decoding site are impaired. Remarkably, overexpression of eIF3g/TIF35 suppresses the leaky scanning and growth defects most probably by preventing these aberrant PICs to form. Leaky scanning is also partially suppressed by eIF1, one of the key regulators of AUG recognition, and its mutant sui1(G107R) but the mechanism differs. We conclude that the C-terminus of eIF3b/PRT1 orchestrates co-operative recruitment of eIF3i/TIF34 and eIF3g/TIF35 to the 40S subunit for a stable and proper assembly of 48S pre-initiation complexes necessary for stringent AUG recognition on mRNAs.

A'-form RNA helices are required for cytoplasmic mRNA transport in Drosophila.

Microtubule-based mRNA transport is widely used to restrict protein expression to specific regions in the cell and has important roles in defining cell polarity and axis determination as well as in neuronal function. However, the structural basis of recognition of cis-acting mRNA localization signals by motor complexes is poorly understood. We have used NMR spectroscopy to describe the first tertiary structure to our knowledge of an RNA element responsible for mRNA transport. The Drosophila melanogaster fs(1)K10 signal, which mediates transport by the dynein motor, forms a stem loop with two double-stranded RNA helices adopting an unusual A'-form conformation with widened major grooves reminiscent of those in B-form DNA. Structure determination of four mutant RNAs and extensive functional assays in Drosophila embryos indicate that the two spatially registered A'-form helices represent critical recognition sites for the transport machinery. Our study provides insights into the basis for RNA cargo recognition and reveals a key biological function encoded by A'-form RNA conformation.

Rapid, nondenaturing RNA purification using weak anion-exchange fast performance liquid chromatography.

We present a simple and fast method for large-scale purification of RNA oligonucleotides suitable for biochemical and structural studies. RNAs are transcribed in vitro with T7 RNA polymerase using linearized plasmid DNA templates. After addition of EDTA, the crude transcription reaction is subjected directly to weak anion-exchange chromatography using DEAE-sepharose to separate the T7 RNA polymerase, unincorporated rNTPs, small abortive transcripts, and the plasmid DNA template from the desired RNA product. The novel method does neither require tedious phenol/chloroform extraction of the T7 RNA polymerase nor denaturation of the RNA, which is desirable especially for larger RNAs. In addition, isotopically labeled rNTPs can be easily recycled from the column flow-through and oligomeric RNA aggregates can be separated from the natively folded monomeric RNA product.

The indispensable N-terminal half of eIF3j/HCR1 cooperates with its structurally conserved binding partner eIF3b/PRT1-RRM and with eIF1A in stringent AUG selection.

Despite recent progress in our understanding of the numerous functions of individual subunits of eukaryotic translation initiation factor (eIF) 3, little is known on the molecular level. Using NMR spectroscopy, we determined the first solution structure of an interaction between eIF3 subunits. We revealed that a conserved tryptophan residue in the human eIF3j N-terminal acidic motif (NTA) is held in the helix alpha1 and loop 5 hydrophobic pocket of the human eIF3b RNA recognition motif (RRM). Mutating the corresponding "pocket" residues in its yeast orthologue reduces cellular growth rate, eliminates eIF3j/HCR1 association with eIF3b/PRT1 in vitro and in vivo, affects 40S occupancy of eIF3, and produces a leaky scanning defect indicative of a deregulation of the AUG selection process. Unexpectedly, we found that the N-terminal half of eIF3j/HCR1 containing the NTA is indispensable and sufficient for wild-type growth of yeast cells. Furthermore, we demonstrate that deletion of either j/HCR1 or its N-terminal half only, or mutation of the key tryptophan residues results in the severe leaky scanning phenotype partially suppressible by overexpressed eIF1A, which is thought to stabilize properly formed preinitiation complexes at the correct start codon. These findings indicate that eIF3j/HCR1 remains associated with the scanning preinitiation complexes and does not dissociate from the small ribosomal subunit upon mRNA recruitment, as previously believed. Finally, we provide further support for earlier mapping of the ribosomal binding site for human eIF3j by identifying specific interactions of eIF3j/HCR1 with small ribosomal proteins RPS2 and RPS23 located in the vicinity of the mRNA entry channel. Taken together, we propose that eIF3j/HCR1 closely cooperates with the eIF3b/PRT1 RRM and eIF1A on the ribosome to ensure proper formation of the scanning-arrested conformation required for stringent AUG recognition.

Conserved functional domains and a novel tertiary interaction near the pseudoknot drive translational activity of hepatitis C virus and hepatitis C virus-like internal ribosome entry sites.

The translational activity of the hepatitis C virus (HCV) internal ribosome entry site (IRES) and other HCV-like IRES RNAs depends on structured RNA elements in domains II and III, which serve to recruit the ribosomal 40S subunit, eukaryotic initiation factor (eIF) 3 and the ternary eIF2/Met-tRNA(i)(Met)/GTP complex and subsequently domain II assists subunit joining. Porcine teschovirus-1 talfan (PTV-1) is a member of the Picornaviridae family, with a predicted HCV-like secondary structure, but only stem-loops IIId and IIIe in the 40S-binding domain display significant sequence conservation with the HCV IRES. Here, we use chemical probing to show that interaction sites with the 40S subunit and eIF3 are conserved between HCV and HCV-like IRESs. In addition, we reveal the functional role of a strictly conserved co-variation between a purine-purine mismatch near the pseudoknot (A-A/G) and the loop sequence of domain IIIe (GAU/CA). These nucleotides are involved in a tertiary interaction, which serves to stabilize the pseudoknot structure and correlates with translational efficiency in both the PTV-1 and HCV IRES. Our data demonstrate conservation of functional domains in HCV and HCV-like IRESs including a more complex structure surrounding the pseudoknot than previously assumed.

Large-scale native preparation of in vitro transcribed RNA.

Biophysical studies of RNA require concentrated samples that are chemically and structurally homogeneous. Historically, the most widely used methods for preparing these samples involve in vitro transcription, denaturation of the RNA, purification based on size, and subsequent refolding. These methods are useful but are inherently slow and do not guarantee that the RNA is properly folded. Possible mis-folding is of particular concern with large, complexly folded RNAs. To address these problems, we have developed methods for purifying in vitro transcribed RNAs in their native, folded states. These methods also have the advantage of being rapid and readily scaled to virtually any size RNA or transcription amount. Two methods are presented: the first is an affinity chromatography approach and the second is a weak ion-exchange chromatography approach. Both use equipment and materials readily available to almost any lab and hence should provide flexibility for those seeking alternate approaches to large-scale purification of RNA in the folded state.

Structure and function of HCV IRES domains.

The HCV IRES is a highly structured RNA which mediates cap-independent translation initiation in higher eukaryotes. This function is encoded in conserved structural motifs in the two major domains of HCV and HCV-like IRESs, which play crucial and distinct roles along the initiation pathway. In this review, I discuss structural features of IRES domains and how these RNA motifs function as RNA-based initiation factors to form 48S initiation complexes and 80S ribosomes with only a subset of canonical, protein-based eukaryotic initiation factors.

A practical approach to isolate 48S complexes: affinity purification and analyses.

In vitro assembly of eukaryotic translation initiation complexes requires purification of ribosomal subunits, eukaryotic initiation factors, and initiator tRNA from natural sources and therefore yields only limited material for functional and structural studies. In this chapter, we describe a robust, affinity chromatography-based method for the isolation of eukaryotic 48S initiation complexes from rabbit reticulocyte lysate (RRL). Both canonical and internal ribosome entry site (IRES)-containing mRNAs labeled with a streptomycin aptamer sequence at the 3' end can be used to purify milligram quantities of 48S particles in a simple, two-step procedure. The 48S complexes purified with this method are properly assembled at the initiation codon, contain the expected RNA and protein components in a 1:1 stoichiometry, and are functional intermediates along the initiation pathway.

Preparation of large RNA oligonucleotides with complementary isotope-labeled segments for NMR structural studies.

RNA structure determination by solution NMR spectroscopy is often restricted to small RNAs (<15 kDa) owing to the problem of chemical shift degeneracy. A fruitful coupling of novel NMR techniques with segmental RNA labeling methodologies could be a powerful tool to overcome the molecular mass limitation of RNA NMR spectroscopy. Herein, we describe a time- and cost-effective procedure to prepare and purify segmentally labeled large RNAs. Two sets of RNA fragments with complementary labeling schemes, such as one fragment (13)C- and the other (15)N-labeled, are prepared by in vitro transcription from a single plasmid DNA. The desired RNA fragments are excised from the primary transcript by two cis-acting hammerhead ribozymes, yielding the required engineered ends for subsequent, complementary ligation. The resulting RNA oligonucleotides display NMR spectra with greatly reduced resonance overlap and thus enable NMR studies of smaller labeled RNA segments within the native context of a large RNA. The procedure is expected to take 3-4 weeks to implement.

HCV and CSFV IRES domain II mediate eIF2 release during 80S ribosome assembly.

Internal ribosome entry site (IRES) RNAs from the hepatitis C virus (HCV) and classical swine fever virus (CSFV) coordinate cap-independent assembly of eukaryotic 48S initiation complexes, consisting of the 40S ribosomal subunit, eukaryotic initiation factor (eIF) 3 and the eIF2/GTP/Met-tRNA(i)(Met) ternary complex. Here, we report that these IRESes also play a functional role during 80S ribosome assembly downstream of 48S complex formation, in promoting eIF5-induced GTP hydrolysis and eIF2/GDP release from the initiation complex. We show that this function is encoded in their independently folded IRES domain II and that it depends both on its characteristic bent conformation and two conserved RNA motifs, an apical hairpin loop and a loop E. Our data suggest a general mode of subunit joining in HCV and HCV-like IRESes.

Structure of eIF3b RNA recognition motif and its interaction with eIF3j: structural insights into the recruitment of eIF3b to the 40 S ribosomal subunit.

Mammalian eIF3 is a 700-kDa multiprotein complex essential for initiation of protein synthesis in eukaryotic cells. It consists of 13 subunits (eIF3a to -m), among which eIF3b serves as a major scaffolding protein. Here we report the solution structure of the N-terminal RNA recognition motif of human eIF3b (eIF3b-RRM) determined by NMR spectroscopy. The structure reveals a noncanonical RRM with a negatively charged surface in the beta-sheet area contradictory with potential RNA binding activity. Instead, eIF3j, which is required for stable 40 S ribosome binding of the eIF3 complex, specifically binds to the rear alpha-helices of the eIF3b-RRM, opposite to its beta-sheet surface. Moreover, we identify that an N-terminal 69-amino acid peptide of eIF3j is sufficient for binding to eIF3b-RRM and that this interaction is essential for eIF3b-RRM recruitment to the 40 S ribosomal subunit. Our results provide the first structure of an important subdomain of a core eIF3 subunit and detailed insights into protein-protein interactions between two eIF3 subunits required for stable eIF3 recruitment to the 40 S subunit.