Structures of B. subtilis Maturation RNases Captured on 50S Ribosome with Pre-rRNAs.

The pathways for ribosomal RNA (rRNA) maturation diverge greatly among the domains of life. In the Gram-positive model bacterium, Bacillus subtilis, the final maturation steps of the two large ribosomal subunit (50S) rRNAs, 23S and 5S pre-rRNAs, are catalyzed by the double-strand specific ribonucleases (RNases) Mini-RNase III and RNase M5, respectively. Here we present a protocol that allowed us to solve the 3.0 and 3.1 Å resolution cryoelectron microscopy structures of these RNases poised to cleave their pre-rRNA substrates within the B. subtilis 50S particle. These data provide the first structural insights into rRNA maturation in bacteria by revealing how these RNases recognize and process double-stranded pre-rRNA. Our structures further uncover how specific ribosomal proteins act as chaperones to correctly fold the pre-rRNA substrates and, for Mini-III, anchor the RNase to the ribosome. These r-proteins thereby serve a quality-control function in the process from accurate ribosome assembly to rRNA processing.

Advances in the Structural and Functional Understanding of m1A RNA Modification.

ConspectusRNA modification is a co- or post-transcriptional process by which specific nucleotides are chemically altered by enzymes after their initial incorporation into the RNA chain, expanding the chemical and functional diversity of RNAs. Our understanding of RNA modifications has changed dramatically in recent years. In the past decade, RNA methyltransferases (MTases) have been highlighted in numerous clinical studies and disease models, modifications have been found to be dynamically regulated by demodification enzymes, and significant technological advances have been made in the fields of RNA sequencing, mass spectrometry, and structural biology. Among RNAs, transfer RNAs (tRNAs) exhibit the greatest diversity and density of post-transcriptional modifications, which allow for potential cross-talks and regulation during their incorporation. N1-methyladenosine (m1A) modification is found in tRNAs at positions 9, 14, 16, 22, 57, and 58, depending on the tRNA and organism.Our laboratory has used and developed a large panel of tools to decipher the different mechanisms used by m1A tRNA MTases to recognize and methylate tRNA. We have solved the structures of TrmI from Thermus thermophilus (m1A58), TrmK from Bacillus subtilis (m1A22), and human TRMT10C (m1A9). These MTases do not share the same structure or organization to recognize tRNAs, but they all modify an adenosine, forming a non-Watson-Crick (WC) interaction. For TrmK, nuclear magnetic resonance (NMR) chemical shift mapping of the binding interface between TrmK and tRNASer was invaluable to build a TrmK/tRNA model, where both domains of TrmK participate in the binding of a full-length L-shaped tRNA and where the non-WC purine 13-A22 base pair positions the A22 N1-atom close to the methyl of the S-adenosyl-l-methionine (SAM) TrmK cofactor. For TRMT10C, cryoEM structures showed the MTase poised to N1-methylate A9 or G9 in tRNA and revealed different steps of tRNA maturation, where TRMT10C acts as a tRNA binding platform for sequential docking of each maturation enzyme. This work confers a role for TRMT10C in tRNA quality control and provides a framework to understand the link between mitochondrial tRNA maturation dysfunction and diseases.Methods to directly detect the incorporation of modifications during tRNA biosynthesis are rare and do not provide easy access to the temporality of their introduction. To this end, we have introduced time-resolved NMR to monitor tRNA maturation in the cellular environment. Combined with genetic and biochemical approaches involving the synthesis of specifically modified tRNAs, our methodology revealed that some modifications are incorporated in a defined sequential order, controlled by cross-talks between modification events. In particular, a strong modification circuit, namely Ψ55 → m5U54 → m1A58, controls the modification process in the T-arm of yeast elongator tRNAs. Conversely, we showed that m1A58 is efficiently introduced on unmodified initiator tRNAiMet without the need of any prior modification. Two distinct pathways are therefore followed for m1A58 incorporation in elongator and initiator tRNAs.We are undoubtedly entering an exciting period for the elucidation of the functions of RNA modifications and the intricate mechanisms by which modification enzymes identify and alter their RNA substrates. These are promising directions for the field of epitranscriptomics.

Different modification pathways for m1A58 incorporation in yeast elongator and initiator tRNAs.

As essential components of the protein synthesis machinery, tRNAs undergo a tightly controlled biogenesis process, which include the incorporation of numerous posttranscriptional modifications. Defects in these tRNA maturation steps may lead to the degradation of hypomodified tRNAs by the rapid tRNA decay (RTD) and nuclear surveillance pathways. We previously identified m1A58 as a late modification introduced after modifications Ψ55 and T54 in yeast elongator tRNAPhe. However, previous reports suggested that m1A58 is introduced early during the tRNA modification process, in particular on primary transcripts of initiator tRNAiMet, which prevents its degradation by RNA decay pathways. Here, aiming to reconcile this apparent inconsistency on the temporality of m1A58 incorporation, we examined its introduction into yeast elongator and initiator tRNAs. We used specifically modified tRNAs to report on the molecular aspects controlling the Ψ55 → T54 → m1A58 modification circuit in elongator tRNAs. We also show that m1A58 is efficiently introduced on unmodified tRNAiMet, and does not depend on prior modifications. Finally, we show that m1A58 has major effects on the structural properties of initiator tRNAiMet, so that the tRNA elbow structure is only properly assembled when this modification is present. This observation provides a structural explanation for the degradation of hypomodified tRNAiMet lacking m1A58 by the nuclear surveillance and RTD pathways.

Synthesis of RNA-cofactor conjugates and structural exploration of RNA recognition by an m6A RNA methyltransferase.

Chemical synthesis of RNA conjugates has opened new strategies to study enzymatic mechanisms in RNA biology. To gain insights into poorly understood RNA nucleotide methylation processes, we developed a new method to synthesize RNA-conjugates for the study of RNA recognition and methyl-transfer mechanisms of SAM-dependent m6A RNA methyltransferases. These RNA conjugates contain a SAM cofactor analogue connected at the N6-atom of an adenosine within dinucleotides, a trinucleotide or a 13mer RNA. Our chemical route is chemo- and regio-selective and allows flexible modification of the RNA length and sequence. These compounds were used in crystallization assays with RlmJ, a bacterial m6A rRNA methyltransferase. Two crystal structures of RlmJ in complex with RNA-SAM conjugates were solved and revealed the RNA-specific recognition elements used by RlmJ to clamp the RNA substrate in its active site. From these structures, a model of a trinucleotide bound in the RlmJ active site could be built and validated by methyltransferase assays on RlmJ mutants. The methyl transfer by RlmJ could also be deduced. This study therefore shows that RNA-cofactor conjugates are potent molecular tools to explore the active site of RNA modification enzymes.

Instrumental analysis of RNA modifications.

Organisms from all domains of life invest a substantial amount of energy for the introduction of RNA modifications into nearly all transcripts studied to date. Instrumental analysis of RNA can focus on the modified residues and reveal the function of these epitranscriptomic marks. Here, we will review recent advances and breakthroughs achieved by NMR spectroscopy, sequencing, and mass spectrometry of the epitranscriptome.

A comprehensive review of m6A/m6Am RNA methyltransferase structures.

Gene expression is regulated at many levels including co- or post-transcriptionally, where chemical modifications are added to RNA on riboses and bases. Expression control via RNA modifications has been termed 'epitranscriptomics' to keep with the related 'epigenomics' for DNA modification. One such RNA modification is the N6-methylation found on adenosine (m6A) and 2'-O-methyladenosine (m6Am) in most types of RNA. The N6-methylation can affect the fold, stability, degradation and cellular interaction(s) of the modified RNA, implicating it in processes such as splicing, translation, export and decay. The multiple roles played by this modification explains why m6A misregulation is connected to multiple human cancers. The m6A/m6Am writer enzymes are RNA methyltransferases (MTases). Structures are available for functionally characterized m6A RNA MTases from human (m6A mRNA, m6A snRNA, m6A rRNA and m6Am mRNA MTases), zebrafish (m6Am mRNA MTase) and bacteria (m6A rRNA MTase). For each of these MTases, we describe their overall domain organization, the active site architecture and the substrate binding. We identify areas that remain to be investigated, propose yet unexplored routes for structural characterization of MTase:substrate complexes, and highlight common structural elements that should be described for future m6A/m6Am RNA MTase structures.

Structural maturation of the HIV-1 RNA 5' untranslated region by Pr55Gag and its maturation products.

Maturation of the HIV-1 viral particles shortly after budding is required for infectivity. During this process, the Pr55Gag precursor undergoes a cascade of proteolytic cleavages, and whilst the structural rearrangements of the viral proteins are well understood, the concomitant maturation of the genomic RNA (gRNA) structure is unexplored, despite evidence that it is required for infectivity. To get insight into this process, we systematically analysed the interactions between Pr55Gag or its maturation products (NCp15, NCp9 and NCp7) and the 5' gRNA region and their structural consequences, in vitro. We show that Pr55Gag and its maturation products mostly bind at different RNA sites and with different contributions of their two zinc knuckle domains. Importantly, these proteins have different transient and permanent effects on the RNA structure, the late NCp9 and NCp7 inducing dramatic structural rearrangements. Altogether, our results reveal the distinct contributions of the different Pr55Gag maturation products on the gRNA structural maturation.

The nucleic acid chaperone activity of the HIV-1 Gag polyprotein is boosted by its cellular partner RPL7: a kinetic study.

The HIV-1 Gag protein playing a key role in HIV-1 viral assembly has recently been shown to interact through its nucleocapsid domain with the ribosomal protein L7 (RPL7) that acts as a cellular co-factor promoting Gag's nucleic acid (NA) chaperone activity. To further understand how the two proteins act together, we examined their mechanism individually and in concert to promote the annealing between dTAR, the DNA version of the viral transactivation element and its complementary cTAR sequence, taken as model HIV-1 sequences. Gag alone or complexed with RPL7 was found to act as a NA chaperone that destabilizes cTAR stem-loop and promotes its annealing with dTAR through the stem ends via a two-step pathway. In contrast, RPL7 alone acts as a NA annealer that through its NA aggregating properties promotes cTAR/dTAR annealing via two parallel pathways. Remarkably, in contrast to the isolated proteins, their complex promoted efficiently the annealing of cTAR with highly stable dTAR mutants. This was confirmed by the RPL7-promoted boost of the physiologically relevant Gag-chaperoned annealing of (+)PBS RNA to the highly stable tRNALys3 primer, favoring the notion that Gag recruits RPL7 to overcome major roadblocks in viral assembly.

Functional diversification in the Nudix hydrolase gene family drives sesquiterpene biosynthesis in Rosa × wichurana.

Roses use a non-canonical pathway involving a Nudix hydrolase, RhNUDX1, to synthesize their monoterpenes, especially geraniol. Here we report the characterization of another expressed NUDX1 gene from the rose cultivar Rosa x wichurana, RwNUDX1-2. In order to study the function of the RwNUDX1-2 protein, we analyzed the volatile profiles of an F1 progeny generated by crossing R. chinensis cv. 'Old Blush' with R. x wichurana. A correlation test of the volatilomes with gene expression data revealed that RwNUDX1-2 is involved in the biosynthesis of a group of sesquiterpenoids, especially E,E-farnesol, in addition to other sesquiterpenes. In vitro enzyme assays and heterologous in planta functional characterization of the RwNUDX1-2 gene corroborated this result. A quantitative trait locus (QTL) analysis was performed using the data of E,E-farnesol contents in the progeny and a genetic map was constructed based on gene markers. The RwNUDX1-2 gene co-localized with the QTL for E,E-farnesol content, thereby confirming its function in sesquiterpenoid biosynthesis in R. x wichurana. Finally, in order to understand the structural bases for the substrate specificity of rose NUDX proteins, the RhNUDX1 protein was crystallized, and its structure was refined to 1.7 Å. By molecular modeling of different rose NUDX1 protein complexes with their respective substrates, a structural basis for substrate discrimination by rose NUDX1 proteins is proposed.

Structural studies of RNase M5 reveal two-metal-ion supported two-step dsRNA cleavage for 5S rRNA maturation.

All species transcribe ribosomal RNA in an immature form that requires several enzymes for processing into mature rRNA. The number and types of enzymes utilized for these processes vary greatly between different species. In low G + C Gram-positive bacteria including Bacillus subtilis and Geobacillus stearothermophilus, the endoribonuclease (RNase) M5 performs the final step in 5S rRNA maturation, by removing the 3'- and 5'-extensions from precursor (pre) 5S rRNA. This cleavage activity requires initial complex formation between the pre-rRNA and a ribosomal protein, uL18, making the full M5 substrate a ribonucleoprotein particle (RNP). M5 contains a catalytic N-terminal Toprim domain and an RNA-binding C-terminal domain, respectively, shown to assist in processing and binding of the RNP. Here, we present structural data that show how two Mg2+ ions are accommodated in the active site pocket of the catalytic Toprim domain and investigate the importance of these ions for catalysis. We further perform solution studies that support the previously proposed 3'-before-5' order of removal of the pre-5S rRNA extensions and map the corresponding M5 structural rearrangements during catalysis.

Structural characterization of B. subtilis m1A22 tRNA methyltransferase TrmK: insights into tRNA recognition.

1-Methyladenosine (m1A) is a modified nucleoside found at positions 9, 14, 22 and 58 of tRNAs, which arises from the transfer of a methyl group onto the N1-atom of adenosine. The yqfN gene of Bacillus subtilis encodes the methyltransferase TrmK (BsTrmK) responsible for the formation of m1A22 in tRNA. Here, we show that BsTrmK displays a broad substrate specificity, and methylates seven out of eight tRNA isoacceptor families of B. subtilis bearing an A22. In addition to a non-Watson-Crick base-pair between the target A22 and a purine at position 13, the formation of m1A22 by BsTrmK requires a full-length tRNA with intact tRNA elbow and anticodon stem. We solved the crystal structure of BsTrmK showing an N-terminal catalytic domain harbouring the typical Rossmann-like fold of Class-I methyltransferases and a C-terminal coiled-coil domain. We used NMR chemical shift mapping to drive the docking of BstRNASer to BsTrmK in complex with its methyl-donor cofactor S-adenosyl-L-methionine (SAM). In this model, validated by methyltransferase activity assays on BsTrmK mutants, both domains of BsTrmK participate in tRNA binding. BsTrmK recognises tRNA with very few structural changes in both partner, the non-Watson-Crick R13-A22 base-pair positioning the A22 N1-atom close to the SAM methyl group.

Molecular basis for transfer RNA recognition by the double-stranded RNA-binding domain of human dihydrouridine synthase 2.

Double stranded RNA-binding domain (dsRBD) is a ubiquitous domain specialized in the recognition of double-stranded RNAs (dsRNAs). Present in many proteins and enzymes involved in various functional roles of RNA metabolism, including RNA splicing, editing, and transport, dsRBD generally binds to RNAs that lack complex structures. However, this belief has recently been challenged by the discovery of a dsRBD serving as a major tRNA binding module for human dihydrouridine synthase 2 (hDus2), a flavoenzyme that catalyzes synthesis of dihydrouridine within the complex elbow structure of tRNA. We here unveil the molecular mechanism by which hDus2 dsRBD recognizes a tRNA ligand. By solving the crystal structure of this dsRBD in complex with a dsRNA together with extensive characterizations of its interaction with tRNA using mutagenesis, NMR and SAXS, we establish that while hDus2 dsRBD retains a conventional dsRNA recognition capability, the presence of an N-terminal extension appended to the canonical domain provides additional residues for binding tRNA in a structure-specific mode of action. Our results support that this extension represents a feature by which the dsRBD specializes in tRNA biology and more broadly highlight the importance of structural appendages to canonical domains in promoting the emergence of functional diversity.

Modulation of the HIV nucleocapsid dynamics finely tunes its RNA-binding properties during virion genesis.

During HIV-1 assembly and budding, Gag protein, in particular the C-terminal domain containing the nucleocapsid domain (NCd), p1 and p6, is the site of numerous interactions with viral and cellular factors. Most in vitro studies of Gag have used constructs lacking p1 and p6. Here, using NMR spectroscopy, we show that the p1-p6 region of Gag (NCp15) is largely disordered, but interacts transiently with the NCd. These interactions modify the dynamic properties of the NCd. Indeed, using isothermal titration calorimetry (ITC), we have measured a higher entropic penalty to RNA-binding for the NCd precursor, NCp15, than for the mature form, NCp7, which lacks p1 and p6. We propose that during assembly and budding of virions, concomitant with Gag oligomerization, transient interactions between NCd and p1-p6 become salient and responsible for (i) a higher level of structuration of p6, which favours recruitment of budding partners; and (ii) a higher entropic penalty to RNA-binding at specific sites that favours non-specific binding of NCd at multiple sites on the genomic RNA (gRNA). The contributions of p6 and p1 are sequentially removed via proteolysis during Gag maturation such that the RNA-binding specificity of the mature protein is governed by the properties of NCd.

Conformational Stability Adaptation of a Double-Stranded RNA-Binding Domain to Transfer RNA Ligand.

The double-stranded RNA-binding domain (dsRBD) is a broadly distributed domain among RNA-maturing enzymes. Although this domain recognizes dsRNA's structures via a conserved canonical structure adopting an α1-ß1ß2ß3-α2 topology, several dsRBDs can accommodate discrete structural extensions expanding further their functional repertoire. How these structural elements engage cooperative communications with the canonical structure and how they contribute to the dsRBD's overall folding are poorly understood. Here, we addressed these issues using the dsRBD of human dihydrouridine synthase-2 (hDus2) (hDus2-dsRBD) as a model. This dsRBD harbors N- and C-terminal extensions, the former being directly involved in the recognition of tRNA substrate of hDus2. These extensions engage residues that form a long-range hydrophobic network (LHN) outside the RNA-binding interface. We show by coarse-grain Brownian dynamics that the Nt-extension and its residues F359 and Y364 rigidify the major folding nucleus of the canonical structure via an indirect effect. hDus2-dsRBD unfolds following a two-state cooperative model, whereas both F359A and Y364A mutants, designed to destabilize this LHN, unfold irreversibly. Structural and computational analyses show that these mutants are unstable due to an increase in the dynamics of the two extensions favoring solvent exposure of α2-helix and weakening the main folding nucleus rigidity. This LHN appears essential for maintaining a thermodynamic stability of the overall system and eventually a functional conformation for tRNA recognition. Altogether, our findings suggest that functional adaptability of extended dsRBDs is promoted by a cooperative hydrophobic coupling between the extensions acting as effectors and the folding nucleus of the canonical structure.

Benefits of stable isotope labeling in RNA analysis.

RNAs are key players in life as they connect the genetic code (DNA) with all cellular processes dominated by proteins. They contain a variety of chemical modifications and many RNAs fold into complex structures. Here, we review recent progress in the analysis of RNA modification and structure on the basis of stable isotope labeling techniques. Mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy are the key tools and many breakthrough developments were made possible by the analysis of stable isotope labeled RNA. Therefore, we discuss current stable isotope labeling techniques such as metabolic labeling, enzymatic labeling and chemical synthesis. RNA structure analysis by NMR is challenging due to two major problems that become even more salient when the size of the RNA increases, namely chemical shift overlaps and line broadening leading to complete signal loss. Several isotope labeling strategies have been developed to provide solutions to these major issues, such as deuteration, segmental isotope labeling or site-specific labeling. Quantification of modified nucleosides in RNA by MS is only possible through the application of stable isotope labeled internal standards. With nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS), it is now possible to analyze the dynamic processes of post-transcriptional RNA modification and demodification. The trend, in both NMR and MS RNA analytics, is without doubt shifting from the analysis of snapshot moments towards the development and application of tools capable of analyzing the dynamics of RNA structure and modification profiles.

To be or not to be modified: Miscellaneous aspects influencing nucleotide modifications in tRNAs.

Transfer RNAs (tRNAs) are essential components of the cellular protein synthesis machineries, but are also implicated in many roles outside translation. To become functional, tRNAs, initially transcribed as longer precursor tRNAs, undergo a tightly controlled biogenesis process comprising the maturation of their extremities, removal of intronic sequences if present, addition of the 3'-CCA amino-acid accepting sequence, and aminoacylation. In addition, the most impressive feature of tRNA biogenesis consists in the incorporation of a large number of posttranscriptional chemical modifications along its sequence. The chemical nature of these modifications is highly diverse, with more than hundred different modifications identified in tRNAs to date. All functions of tRNAs in cells are controlled and modulated by modifications, making the understanding of the mechanisms that determine and influence nucleotide modifications in tRNAs an essential point in tRNA biology. This review describes the different aspects that determine whether a certain position in a tRNA molecule is modified or not. We describe how sequence and structural determinants, as well as the presence of prior modifications control modification processes. We also describe how environmental factors and cellular stresses influence the level and/or the nature of certain modifications introduced in tRNAs, and report situations where these dynamic modulations of tRNA modification levels are regulated by active demodification processes. © 2019 IUBMB Life, 71(8):1126-1140, 2019.

Bisubstrate analogues as structural tools to investigate m6A methyltransferase active sites.

RNA methyltransferases (MTases) catalyse the transfer of a methyl group to their RNA substrates using most-often S-adenosyl-L-methionine (SAM) as cofactor. Only few RNA-bound MTases structures are currently available due to the difficulties in crystallising RNA:protein complexes. The lack of complex structures results in poorly understood RNA recognition patterns and methylation reaction mechanisms. On the contrary, many cofactor-bound MTase structures are available, resulting in well-understood protein:cofactor recognition, that can guide the design of bisubstrate analogues that mimic the state at which both the substrate and the cofactor is bound. Such bisubstrate analogues were recently synthesized for proteins monomethylating the N6-atom of adenine (m6A). These proteins include, amongst others, RlmJ in E. coli and METLL3:METT14 and METTL16 in human. As a proof-of-concept, we here test the ability of the bisubstrate analogues to mimic the substrate:cofactor bound state during catalysis by studying their binding to RlmJ using differential scanning fluorimetry, isothermal titration calorimetry and X-ray crystallography. We find that the methylated adenine base binds in the correct pocket, and thus these analogues could potentially be used broadly to study the RNA recognition and catalytic mechanism of m6A MTases. Two bisubstrate analogues bind RlmJ with micro-molar affinity, and could serve as starting scaffolds for inhibitor design against m6A RNA MTases. The same analogues cause changes in the melting temperature of the m1A RNA MTase, TrmK, indicating non-selective protein:compound complex formation. Thus, optimization of these molecular scaffolds for m6A RNA MTase inhibition should aim to increase selectivity, as well as affinity.

Insights into the mechanisms of RNA secondary structure destabilization by the HIV-1 nucleocapsid protein.

The mature HIV-1 nucleocapsid protein NCp7 (NC) plays a key role in reverse transcription facilitating the two obligatory strand transfers. Several properties contribute to its efficient chaperon activity: preferential binding to single-stranded regions, nucleic acid aggregation, helix destabilization, and rapid dissociation from nucleic acids. However, little is known about the relationships between these different properties, which are complicated by the ability of the protein to recognize particular HIV-1 stem-loops, such as SL1, SL2, and SL3, with high affinity and without destabilizing them. These latter properties are important in the context of genome packaging, during which NC is part of the Gag precursor. We used NMR to investigate destabilization of the full-length TAR (trans activating response element) RNA by NC, which is involved in the first strand transfer step of reverse transcription. NC was used at a low protein:nucleotide (nt) ratio of 1:59 in these experiments. NMR data for the imino protons of TAR identified most of the base pairs destabilized by NC. These base pairs were adjacent to the loops in the upper part of the TAR hairpin rather than randomly distributed. Gel retardation assays showed that conversion from the initial TAR-cTAR complex to the fully annealed form occurred much more slowly at the 1:59 ratio than at the higher ratios classically used. Nevertheless, NC significantly accelerated the formation of the initial complex at a ratio of 1:59.

The NC domain of HIV-1 Gag contributes to the interaction of Gag with TSG101.

BACKGROUND: HIV-1 Gag polyprotein orchestrates the assembly of viral particles. Its C-terminus consists of the nucleocapsid (NC) domain that interacts with RNA, and the p6 domain containing the PTAP motif that binds the cellular ESCRT factor TSG101 and ALIX. Deletion of the NC domain of Gag (GagNC) results in defective Gag assembly, a decrease in virus production and, thus probably affects recruitment of the ESCRT machinery. To investigate the role of GagNC in this recruitment, we analysed its impact on TSG101 and ALIX localisations and interactions in cells expressing Gag. METHODS: Cells expressing mCherry-Gag or derivatives, alone or together with eGFP-TSG101 or eGFP-ALIX, were analysed by confocal microscopy and FLIM-FRET. Chemical shift mapping between TSG101-UEV motif and Gag C-terminus was performed by NMR. RESULTS: We show that deletion of NC or of its two zinc fingers decreases the amount of Gag-TSG101 interacting complexes in cells. These findings are supported by NMR data showing chemical shift perturbations in the NC domain in- and outside - of the zinc finger elements upon TSG101 binding. The NMR data further identify a large stretch of amino acids within the p6 domain directly interacting with TSG101. CONCLUSION: The NC zinc fingers and p6 domain of Gag participate in the formation of the Gag-TSG101 complex and in its cellular localisation. GENERAL SIGNIFICANCE: This study illustrates that the NC and p6 domains cooperate in the interaction with TSG101 during HIV-1 budding. In addition, details on the Gag-TSG101 complex were obtained by combining two high resolution biophysical techniques.

Bacillus subtilis RNA deprotection enzyme RppH recognizes guanosine in the second position of its substrates.

The initiation of mRNA degradation often requires deprotection of its 5' end. In eukaryotes, the 5'-methylguanosine (cap) structure is principally removed by the Nudix family decapping enzyme Dcp2, yielding a 5'-monophosphorylated RNA that is a substrate for 5' exoribonucleases. In bacteria, the 5'-triphosphate group of primary transcripts is also converted to a 5' monophosphate by a Nudix protein called RNA pyrophosphohydrolase (RppH), allowing access to both endo- and 5' exoribonucleases. Here we present the crystal structures of Bacillus subtilis RppH (BsRppH) bound to GTP and to a triphosphorylated dinucleotide RNA. In contrast to Bdellovibrio bacteriovorus RppH, which recognizes the first nucleotide of its RNA targets, the B. subtilis enzyme has a binding pocket that prefers guanosine residues in the second position of its substrates. The identification of sequence specificity for RppH in an internal position was a highly unexpected result. NMR chemical shift mapping in solution shows that at least three nucleotides are required for unambiguous binding of RNA. Biochemical assays of BsRppH on RNA substrates with single-base-mutation changes in the first four nucleotides confirm the importance of guanosine in position two for optimal enzyme activity. Our experiments highlight important structural and functional differences between BsRppH and the RNA deprotection enzymes of distantly related bacteria.