Structural basis of microRNA biogenesis by Dicer-1 and its partner protein Loqs-PB.

In animals and plants, Dicer enzymes collaborate with double-stranded RNA-binding domain (dsRBD) proteins to convert precursor-microRNAs (pre-miRNAs) into miRNA duplexes. We report six cryo-EM structures of Drosophila Dicer-1 that show how Dicer-1 and its partner Loqs­PB cooperate (1) before binding pre-miRNA, (2) after binding and in a catalytically competent state, (3) after nicking one arm of the pre-miRNA, and (4) following complete dicing and initial product release. Our reconstructions suggest that pre-miRNA binds a rare, open conformation of the Dicer­1⋅Loqs­PB heterodimer. The Dicer-1 dsRBD and three Loqs­PB dsRBDs form a tight belt around the pre-miRNA, distorting the RNA helix to place the scissile phosphodiester bonds in the RNase III active sites. Pre-miRNA cleavage shifts the dsRBDs and partially closes Dicer-1, which may promote product release. Our data suggest a model for how the Dicer­1⋅Loqs­PB complex affects a complete cycle of pre-miRNA recognition, stepwise endonuclease cleavage, and product release.

The translational repressor Glorund uses interchangeable RNA recognition domains to recognize Drosophila nanos.

The Drosophila melanogaster protein Glorund (Glo) represses nanos (nos) translation and uses its quasi-RNA recognition motifs (qRRMs) to recognize both G-tract and structured UA-rich motifs within the nos translational control element (TCE). We showed previously that each of the three qRRMs is multifunctional, capable of binding to G-tract and UA-rich motifs, yet if and how the qRRMs combine to recognize the nos TCE remained unclear. Here we determined solution structures of a nos TCEI_III RNA containing the G-tract and UA-rich motifs. The RNA structure demonstrated that a single qRRM is physically incapable of recognizing both RNA elements simultaneously. In vivo experiments further indicated that any two qRRMs are sufficient to repress nos translation. We probed interactions of Glo qRRMs with TCEI_III RNA using NMR paramagnetic relaxation experiments. Our in vitro and in vivo data support a model whereby tandem Glo qRRMs are indeed multifunctional and interchangeable for recognition of TCE G-tract or UA-rich motifs. This study illustrates how multiple RNA recognition modules within an RNA-binding protein may combine to diversify the RNAs that are recognized and regulated.

Bipartite interaction sites differentially modulate RNA-binding affinity of a protein complex essential for germline stem cell self-renewal.

In C. elegans, PUF proteins promote germline stem cell self-renewal. Their functions hinge on partnerships with two proteins that are redundantly required for stem cell maintenance. Here we focus on understanding how the essential partner protein, LST-1, modulates mRNA regulation by the PUF protein, FBF-2. LST-1 contains two nonidentical sites of interaction with FBF-2, LST-1 A and B. Our crystal structures of complexes of FBF-2, LST-1 A, and RNA visualize how FBF-2 associates with LST-1 A versus LST-1 B. One commonality is that FBF-2 contacts the conserved lysine and leucine side chains in the KxxL motifs in LST-1 A and B. A key difference is that FBF-2 forms unique contacts with regions N- and C-terminal to the KxxL motif. Consequently, LST-1 A does not modulate the RNA-binding affinity of FBF-2, whereas LST-1 B decreases RNA-binding affinity of FBF-2. The N-terminal region of LST-1 B, which binds near the 5' end of RNA elements, is essential to modulate FBF-2 RNA-binding affinity, while the C-terminal residues of LST-1 B contribute strong binding affinity to FBF-2. We conclude that LST-1 has the potential to impact which mRNAs are regulated depending on the precise nature of engagement through its functionally distinct FBF binding sites.

High-resolution structures of the SAMHD1 dGTPase homolog from Leeuwenhoekiella blandensis reveal a novel mechanism of allosteric activation by dATP.

Deoxynucleoside triphosphate (dNTP) triphosphohydrolases (dNTPases) are important enzymes that may perform multiple functions in the cell, including regulating the dNTP pools and contributing to innate immunity against viruses. Among the homologs that are best studied are human sterile alpha motif and HD domain-containing protein 1 (SAMHD1), a tetrameric dNTPase, and the hexameric Escherichia coli dGTPase; however, it is unclear whether these are representative of all dNTPases given their wide distribution throughout life. Here, we investigated a hexameric homolog from the marine bacterium Leeuwenhoekiella blandensis, revealing that it is a dGTPase that is subject to allosteric activation by dATP, specifically. Allosteric regulation mediated solely by dATP represents a novel regulatory feature among dNTPases that may facilitate maintenance of cellular dNTP pools in L. blandensis. We present high-resolution X-ray crystallographic structures (1.80-2.26 Å) in catalytically important conformations as well as cryo-EM structures (2.1-2.7 Å) of the enzyme bound to dGTP and dATP ligands. The structures, the highest resolution cryo-EM structures of any SAMHD1-like dNTPase to date, reveal an intact metal-binding site with the dGTP substrate coordinated to three metal ions. These structural and biochemical data yield insights into the catalytic mechanism and support a conserved catalytic mechanism for the tetrameric and hexameric dNTPase homologs. We conclude that the allosteric activation by dATP appears to rely on structural connectivity between the allosteric and active sites, as opposed to the changes in oligomeric state upon ligand binding used by SAMHD1.

Pentatricopeptide repeats of protein-only RNase P use a distinct mode to recognize conserved bases and structural elements of pre-tRNA.

Pentatricopeptide repeat (PPR) motifs are α-helical structures known for their modular recognition of single-stranded RNA sequences with each motif in a tandem array binding to a single nucleotide. Protein-only RNase P 1 (PRORP1) in Arabidopsis thaliana is an endoribonuclease that uses its PPR domain to recognize precursor tRNAs (pre-tRNAs) as it catalyzes removal of the 5'-leader sequence from pre-tRNAs with its NYN metallonuclease domain. To gain insight into the mechanism by which PRORP1 recognizes tRNA, we determined a crystal structure of the PPR domain in complex with yeast tRNAPhe at 2.85 Å resolution. The PPR domain of PRORP1 bound to the structurally conserved elbow of tRNA and recognized conserved structural features of tRNAs using mechanisms that are different from the established single-stranded RNA recognition mode of PPR motifs. The PRORP1 PPR domain-tRNAPhe structure revealed a conformational change of the PPR domain upon tRNA binding and moreover demonstrated the need for pronounced overall flexibility in the PRORP1 enzyme conformation for substrate recognition and catalysis. The PRORP1 PPR motifs have evolved strategies for protein-tRNA interaction analogous to tRNA recognition by the RNA component of ribonucleoprotein RNase P and other catalytic RNAs, indicating convergence on a common solution for tRNA substrate recognition.

H/ACA snoRNA levels are regulated during stem cell differentiation.

H/ACA small nucleolar RNAs (snoRNAs) guide pseudouridylation as part of a small nucleolar ribonucleoprotein complex (snoRNP). Disruption of H/ACA snoRNA levels in stem cells impairs pluripotency, yet it remains unclear how H/ACA snoRNAs contribute to differentiation. To determine if H/ACA snoRNA levels are dynamic during differentiation, we comprehensively profiled H/ACA snoRNA abundance in multiple murine cell types and during differentiation in three cellular models, including mouse embryonic stem cells and mouse myoblasts. We determined that the profiles of H/ACA snoRNA abundance are cell-type specific, and we identified a subset of snoRNAs that are specifically regulated during differentiation. Additionally, we demonstrated that a decrease in Snora27 abundance upon differentiation corresponds to a decrease in pseudouridylation of its target site within the E-site transfer RNA (tRNA) binding region of the 28S ribosomal RNA (rRNA) in the large ribosomal subunit. Together, these data point toward a potential model in which H/ACA snoRNAs are specifically regulated during differentiation to alter pseudouridylation and fine tune ribosome function.

Post-transcriptional Regulatory Functions of Mammalian Pumilio Proteins.

Mammalian Pumilio proteins, PUM1 and PUM2, are members of the PUF family of sequence-specific RNA-binding proteins. In this review, we explore their mechanisms, regulatory networks, biological functions, and relevance to diseases. Pumilio proteins bind an extensive network of mRNAs and repress protein expression by inhibiting translation and promoting mRNA decay. Opposingly, in certain contexts, they can activate protein expression. Pumilio proteins also regulate noncoding (nc)RNAs. The ncRNA, ncRNA activated by DNA damage (NORAD), can in turn modulate Pumilio activity. Genetic analysis provides new insights into Pumilio protein function. They are essential for growth and development. They control diverse processes, including stem cell fate, and neurological functions, such as behavior and memory formation. Novel findings show that their dysfunction contributes to neurodegeneration, epilepsy, movement disorders, intellectual disability, infertility, and cancer.

Distinct RNA-binding modules in a single PUF protein cooperate to determine RNA specificity.

PUF proteins, named for Drosophila Pumilio (PUM) and Caenorhabditis elegans fem-3-binding factor (FBF), recognize specific sequences in the mRNAs they bind and control. RNA binding by classical PUF proteins is mediated by a characteristic PUM homology domain (PUM-HD). The Puf1 and Puf2 proteins possess a distinct architecture and comprise a highly conserved subfamily among fungal species. Puf1/Puf2 proteins contain two types of RNA-binding domain: a divergent PUM-HD and an RNA recognition motif (RRM). They recognize RNAs containing UAAU motifs, often in clusters. Here, we report a crystal structure of the PUM-HD of a fungal Puf1 in complex with a dual UAAU motif RNA. Each of the two UAAU tetranucleotides are bound by a Puf1 PUM-HD forming a 2:1 protein-to-RNA complex. We also determined crystal structures of the Puf1 RRM domain that identified a dimerization interface. The PUM-HD and RRM domains act in concert to determine RNA-binding specificity: the PUM-HD dictates binding to UAAU, and dimerization of the RRM domain favors binding to dual UAAU motifs rather than a single UAAU. Cooperative action of the RRM and PUM-HD identifies a new mechanism by which multiple RNA-binding modules in a single protein collaborate to create a unique RNA-binding specificity.

U7 snRNP is recruited to histone pre-mRNA in a FLASH-dependent manner by two separate regions of the stem-loop binding protein.

Cleavage of histone pre-mRNAs at the 3' end requires stem-loop binding protein (SLBP) and U7 snRNP that consists of U7 snRNA and a unique Sm ring containing two U7-specific proteins: Lsm10 and Lsm11. Lsm11 interacts with FLASH and together they bring a subset of polyadenylation factors to U7 snRNP, including the CPSF73 endonuclease that cleaves histone pre-mRNA. SLBP binds to a conserved stem-loop structure upstream of the cleavage site and acts by promoting an interaction between the U7 snRNP and a sequence element located downstream from the cleavage site. We show that both human and Drosophila SLBPs stabilize U7 snRNP on histone pre-mRNA via two regions that are not directly involved in recognizing the stem-loop structure: helix B of the RNA binding domain and the C-terminal region that follows the RNA binding domain. Stabilization of U7 snRNP binding to histone pre-mRNA by SLBP requires FLASH but not the polyadenylation factors. Thus, FLASH plays two roles in 3' end processing of histone pre-mRNAs: It interacts with Lsm11 to form a docking platform for the polyadenylation factors, and it cooperates with SLBP to recruit U7 snRNP to histone pre-mRNA.

Structural analysis reveals the flexible C-terminus of Nop15 undergoes rearrangement to recognize a pre-ribosomal RNA folding intermediate.

The RNA recognition motif (RRM) is the most abundant RNA-binding domain in eukaryotes, and it plays versatile roles in RNA metabolism. Despite its abundance, diversity of RRM structure and function is generated by variations on a conserved core. Yeast Nop15 is an RRM protein that is essential for large ribosomal subunit biogenesis. We determined a 2.0 Å crystal structure of Nop15 that reveals a C-terminal α-helical region obscures its canonical RNA-binding surface. Small-angle X-ray scattering, NMR and RNA-binding analyses further reveal that the C-terminal residues of Nop15 are highly flexible, but essential for tight RNA binding. Moreover, comparison with a recently reported cryo-electron microscopy structure indicates that dramatic rearrangement of the C-terminal region of Nop15 in the pre-ribosome exposes the RNA-binding surface to recognize the base of its stem-loop target RNA and extends a newly-formed α helix to the distal loop where it forms protein interactions.

A divergent Pumilio repeat protein family for pre-rRNA processing and mRNA localization.

Pumilio/feminization of XX and XO animals (fem)-3 mRNA-binding factor (PUF) proteins bind sequence specifically to mRNA targets using a single-stranded RNA-binding domain comprising eight Pumilio (PUM) repeats. PUM repeats have now been identified in proteins that function in pre-rRNA processing, including human Puf-A and yeast Puf6. This is a role not previously ascribed to PUF proteins. Here we present crystal structures of human Puf-A that reveal a class of nucleic acid-binding proteins with 11 PUM repeats arranged in an "L"-like shape. In contrast to classical PUF proteins, Puf-A forms sequence-independent interactions with DNA or RNA, mediated by conserved basic residues. We demonstrate that equivalent basic residues in yeast Puf6 are important for RNA binding, pre-rRNA processing, and mRNA localization. Thus, PUM repeats can be assembled into alternative folds that bind to structured nucleic acids in addition to forming canonical eight-repeat crescent-shaped RNA-binding domains found in classical PUF proteins.

Molecular mechanisms for the regulation of histone mRNA stem-loop-binding protein by phosphorylation.

Replication-dependent histone mRNAs end with a conserved stem loop that is recognized by stem-loop-binding protein (SLBP). The minimal RNA-processing domain of SLBP is phosphorylated at an internal threonine, and Drosophila SLBP (dSLBP) also is phosphorylated at four serines in its 18-aa C-terminal tail. We show that phosphorylation of dSLBP increases RNA-binding affinity dramatically, and we use structural and biophysical analyses of dSLBP and a crystal structure of human SLBP phosphorylated on the internal threonine to understand the striking improvement in RNA binding. Together these results suggest that, although the C-terminal tail of dSLBP does not contact the RNA, phosphorylation of the tail promotes SLBP conformations competent for RNA binding and thereby appears to reduce the entropic penalty for the association. Increased negative charge in this C-terminal tail balances positively charged residues, allowing a more compact ensemble of structures in the absence of RNA.

Patterns and plasticity in RNA-protein interactions enable recruitment of multiple proteins through a single site.

mRNA control hinges on the specificity and affinity of proteins for their RNA binding sites. Regulatory proteins must bind their own sites and reject even closely related noncognate sites. In the PUF [Pumilio and fem-3 binding factor (FBF)] family of RNA binding proteins, individual proteins discriminate differences in the length and sequence of binding sites, allowing each PUF to bind a distinct battery of mRNAs. Here, we show that despite these differences, the pattern of RNA interactions is conserved among PUF proteins: the two ends of the PUF protein make critical contacts with the two ends of the RNA sites. Despite this conserved "two-handed" pattern of recognition, the RNA sequence is flexible. Among the binding sites of yeast Puf4p, RNA sequence dictates the pattern in which RNA bases are flipped away from the binding surface of the protein. Small differences in RNA sequence allow new modes of control, recruiting Puf5p in addition to Puf4p to a single site. This embedded information adds a new layer of biological meaning to the connections between RNA targets and PUF proteins.

Divergence of Pumilio/fem-3 mRNA binding factor (PUF) protein specificity through variations in an RNA-binding pocket.

mRNA control networks depend on recognition of specific RNA sequences. Pumilio-fem-3 mRNA binding factor (PUF) RNA-binding proteins achieve that specificity through variations on a conserved scaffold. Saccharomyces cerevisiae Puf3p achieves specificity through an additional binding pocket for a cytosine base upstream of the core RNA recognition site. Here we demonstrate that this chemically simple adaptation is prevalent and contributes to the diversity of RNA specificities among PUF proteins. Bioinformatics analysis shows that mRNAs associated with Caenorhabditis elegans fem-3 mRNA binding factor (FBF)-2 in vivo contain an upstream cytosine required for biological regulation. Crystal structures of FBF-2 and C. elegans PUF-6 reveal binding pockets structurally similar to that of Puf3p, whereas sequence alignments predict a pocket in PUF-11. For Puf3p, FBF-2, PUF-6, and PUF-11, the upstream pockets and a cytosine are required for maximal binding to RNA, but the quantitative impact on binding affinity varies. Furthermore, the position of the upstream cytosine relative to the core PUF recognition site can differ, which in the case of FBF-2 originally masked the identification of this consensus sequence feature. Importantly, other PUF proteins lack the pocket and so do not discriminate upstream bases. A structure-based alignment reveals that these proteins lack key residues that would contact the cytosine, and in some instances, they also present amino acid side chains that interfere with binding. Loss of the pocket requires only substitution of one serine, as appears to have occurred during the evolution of certain fungal species.

Structural basis for an inositol pyrophosphate kinase surmounting phosphate crowding.

Inositol pyrophosphates (such as IP7 and IP8) are multifunctional signaling molecules that regulate diverse cellular activities. Inositol pyrophosphates have 'high-energy' phosphoanhydride bonds, so their enzymatic synthesis requires that a substantial energy barrier to the transition state be overcome. Additionally, inositol pyrophosphate kinases can show stringent ligand specificity, despite the need to accommodate the steric bulk and intense electronegativity of nature's most concentrated three-dimensional array of phosphate groups. Here we examine how these catalytic challenges are met by describing the structure and reaction cycle of an inositol pyrophosphate kinase at the atomic level. We obtained crystal structures of the kinase domain of human PPIP5K2 complexed with nucleotide cofactors and either substrates, product or a MgF(3)(-) transition-state mimic. We describe the enzyme's conformational dynamics, its unprecedented topological presentation of nucleotide and inositol phosphate, and the charge balance that facilitates partly associative in-line phosphoryl transfer.

Intra- and inter-molecular regulation by intrinsically-disordered regions governs PUF protein RNA binding.

PUF proteins are characterized by globular RNA-binding domains. They also interact with partner proteins that modulate their RNA-binding activities. Caenorhabditis elegans PUF protein fem-3 binding factor-2 (FBF-2) partners with intrinsically disordered Lateral Signaling Target-1 (LST-1) to regulate target mRNAs in germline stem cells. Here, we report that an intrinsically disordered region (IDR) at the C-terminus of FBF-2 autoinhibits its RNA-binding affinity by increasing the off rate for RNA binding. Moreover, the FBF-2 C-terminal region interacts with its globular RNA-binding domain at the same site where LST-1 binds. This intramolecular interaction restrains an electronegative cluster of amino acid residues near the 5' end of the bound RNA to inhibit RNA binding. LST-1 binding in place of the FBF-2 C-terminus therefore releases autoinhibition and increases RNA-binding affinity. This regulatory mechanism, driven by IDRs, provides a biochemical and biophysical explanation for the interdependence of FBF-2 and LST-1 in germline stem cell self-renewal.

Specific and modular binding code for cytosine recognition in Pumilio/FBF (PUF) RNA-binding domains.

Pumilio/fem-3 mRNA-binding factor (PUF) proteins possess a recognition code for bases A, U, and G, allowing designed RNA sequence specificity of their modular Pumilio (PUM) repeats. However, recognition side chains in a PUM repeat for cytosine are unknown. Here we report identification of a cytosine-recognition code by screening random amino acid combinations at conserved RNA recognition positions using a yeast three-hybrid system. This C-recognition code is specific and modular as specificity can be transferred to different positions in the RNA recognition sequence. A crystal structure of a modified PUF domain reveals specific contacts between an arginine side chain and the cytosine base. We applied the C-recognition code to design PUF domains that recognize targets with multiple cytosines and to generate engineered splicing factors that modulate alternative splicing. Finally, we identified a divergent yeast PUF protein, Nop9p, that may recognize natural target RNAs with cytosine. This work deepens our understanding of natural PUF protein target recognition and expands the ability to engineer PUF domains to recognize any RNA sequence.

Engineering splicing factors with designed specificities.

Alternative splicing is generally regulated by trans-acting factors that specifically bind pre-mRNA to activate or inhibit the splicing reaction. This regulation is critical for normal gene expression, and dysregulation of splicing is closely associated with human diseases. Here we engineered artificial splicing factors by combining sequence-specific RNA-binding domains of human Pumilio1 with functional domains that regulate splicing. We applied these factors to modulate different types of alternative splicing in selected targets, to examine the activity of effector domains from natural splicing factors and to modulate splicing of an endogenous human gene, Bcl-X, an anticancer target. The designer factor targeted to Bcl-X increased the amount of pro-apoptotic Bcl-xS splice isoform, thus promoting apoptosis and increasing chemosensitivity of cancer cells to common antitumor drugs. Our approach permitted the creation of artificial factors to target virtually any pre-mRNA, providing a strategy to study splicing regulation and to manipulate disease-associated splicing events.

Structural basis for specific recognition of multiple mRNA targets by a PUF regulatory protein.

Caenorhabditis elegans fem-3 binding factor (FBF) is a founding member of the PUMILIO/FBF (PUF) family of mRNA regulatory proteins. It regulates multiple mRNAs critical for stem cell maintenance and germline development. Here, we report crystal structures of FBF in complex with 6 different 9-nt RNA sequences, including elements from 4 natural mRNAs. These structures reveal that FBF binds to conserved bases at positions 1-3 and 7-8. The key specificity determinant of FBF vs. other PUF proteins lies in positions 4-6. In FBF/RNA complexes, these bases stack directly with one another and turn away from the RNA-binding surface. A short region of FBF is sufficient to impart its unique specificity and lies directly opposite the flipped bases. We suggest that this region imposes a flattened curvature on the protein; hence, the requirement for the additional nucleotide. The principles of FBF/RNA recognition suggest a general mechanism by which PUF proteins recognize distinct families of RNAs yet exploit very nearly identical atomic contacts in doing so.

A 5' cytosine binding pocket in Puf3p specifies regulation of mitochondrial mRNAs.

A single regulatory protein can control the fate of many mRNAs with related functions. The Puf3 protein of Saccharomyces cerevisiae is exemplary, as it binds and regulates more than 100 mRNAs that encode proteins with mitochondrial function. Here we elucidate the structural basis of that specificity. To do so, we explore the crystal structures of Puf3p complexes with 2 cognate RNAs. The key determinant of Puf3p specificity is an unusual interaction between a distinctive pocket of the protein with an RNA base outside the "core" PUF-binding site. That interaction dramatically affects binding affinity in vitro and is required for regulation in vivo. The Puf3p structures, combined with those of Puf4p in the same organism, illuminate the structural basis of natural PUF-RNA networks. Yeast Puf3p binds its own RNAs because they possess a -2C and is excluded from those of Puf4p which contain an additional nucleotide in the core-binding site.