Molecular basis of mRNA delivery to the bacterial ribosome.

Protein synthesis begins with the formation of a ribosome-mRNA complex. In bacteria, the 30S ribosomal subunit is recruited to many mRNAs through base pairing with the Shine Dalgarno (SD) sequence and RNA binding by ribosomal protein bS1. Translation can initiate on nascent mRNAs and RNA polymerase (RNAP) can promote recruitment of the pioneering 30S subunit. Here we examined ribosome recruitment to nascent mRNAs using cryo-EM, single-molecule fluorescence co-localization, and in-cell crosslinking mass spectrometry. We show that bS1 delivers the mRNA to the ribosome for SD duplex formation and 30S subunit activation. Additionally, bS1 mediates the stimulation of translation initiation by RNAP. Together, our work provides a mechanistic framework for how the SD duplex, ribosomal proteins and RNAP cooperate in 30S recruitment to mRNAs and establish transcription-translation coupling.

An evolutionarily conserved N-terminal leucine is essential for MX1 GTPase antiviral activity against different families of RNA viruses.

Myxovirus resistance protein 1 (MX1) and MX2 are homologous, dynamin-like large GTPases, induced upon interferon exposure. Human MX1 (HsMX1) is known to inhibit many viruses, including influenza A virus, by likely acting at various steps of their life cycles. Despite decades of studies, the mechanism(s) of action with which MX1 proteins manage to inhibit target viruses is not fully understood. MX1 proteins are mechano-enzymes and share a similar organization to dynamin, with a GTPase domain and a carboxy-terminal stalk domain, connected by a bundle signaling element. These three elements are known to be essential for antiviral activity. HsMX1 has two unstructured regions, the L4 loop, also essential for antiviral activity, and a short amino (N)-terminal region, which greatly varies between MX1 proteins of different species. The role of this N-terminal domain in antiviral activity is not known. Herein, using mutagenesis, imaging, and biochemical approaches, we demonstrate that the N-terminal domain of HsMX1 is essential for antiviral activity against influenza A virus, Vesicular Stomatitis Virus, and La Crosse virus. Furthermore, we pinpoint a highly conserved leucine within this region, which is absolutely crucial for human, mouse, and bat MX1 protein antiviral activity. Importantly, mutation of this leucine does not compromise GTPase activity or oligomerization capabilities but does modify MX1 protein subcellular localization. The discovery of this essential and highly conserved residue defines this region as key for antiviral activity and may reveal insights as to the mechanism(s) of action of MX1 proteins.

Structural insights into RNA-mediated transcription regulation in bacteria.

RNA can regulate its own synthesis without auxiliary proteins. For example, U-rich RNA sequences signal RNA polymerase (RNAP) to pause transcription and are required for transcript release at intrinsic terminators in all kingdoms of life. In contrast, the regulatory RNA putL suppresses pausing and termination in cis. However, how nascent RNA modulates its own synthesis remains largely unknown. We present cryo-EM reconstructions of RNAP captured during transcription of putL variants or an unrelated sequence at a U-rich pause site. Our results suggest how putL suppresses pausing and promotes its synthesis. We demonstrate that transcribing a U-rich sequence, a ubiquitous trigger of intrinsic termination, promotes widening of the RNAP nucleic-acid-binding channel. Widening destabilizes RNAP interactions with DNA and RNA to facilitate transcript dissociation reminiscent of intrinsic transcription termination. Surprisingly, RNAP remains bound to DNA after transcript release. Our results provide the structural framework to understand RNA-mediated intrinsic transcription termination.

Artificial Intelligence and Clinical Decision Support Systems or Automated Interpreters: What Characteristics Are Expected by French General Practitioners?

Development of artificial intelligence (AI) modules should rely on technical progress, but also on users' needs. Our objective is to identify criteria that make a hypothetical AI module desirable for general practitioners (GPs). Method: random selection of 200 French GPs, and paper-based questionnaire. Results: the population was representative. GPs expect AI modules to diagnose or eliminate an urgent pathology for which they are not competent and for which specialists are not available. They also demand interoperability, automated electronic health record integration and facilitated information sharing. GPs would like AI modules to make them save time, simplify some procedures and delegate tasks to the secretary. They expect AI modules to allow them to associate the patient with the care, to reassure him or her, and to personalize the care. Interestingly, GPs would also rely on a machine to cut off abusive requests, such as work stoppages or certificates of convenience.

Transcription factors modulate RNA polymerase conformational equilibrium.

RNA polymerase (RNAP) frequently pauses during the transcription of DNA to RNA to regulate gene expression. Transcription factors NusA and NusG modulate pausing, have opposing roles, but can bind RNAP simultaneously. Here we report cryo-EM reconstructions of Escherichia coli RNAP bound to NusG, or NusA, or both. RNAP conformational changes, referred to as swivelling, correlate with transcriptional pausing. NusA facilitates RNAP swivelling to further increase pausing, while NusG counteracts this role. Their structural effects are consistent with biochemical results on two categories of transcriptional pauses. In addition, the structures suggest a cooperative mechanism of NusA and NusG during Rho-mediated transcription termination. Our results provide a structural rationale for the stochastic nature of pausing and termination and how NusA and NusG can modulate it.

Structural organization and dynamics of FCHo2 docking on membranes.

Clathrin-mediated endocytosis (CME) is a central trafficking pathway in eukaryotic cells regulated by phosphoinositides. The plasma membrane phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) plays an instrumental role in driving CME initiation. The F-BAR domain-only protein 1 and 2 complex (FCHo1/2) is among the early proteins that reach the plasma membrane, but the exact mechanisms triggering its recruitment remain elusive. Here, we show the molecular dynamics of FCHo2 self-assembly on membranes by combining minimal reconstituted in vitro and cellular systems. Our results indicate that PI(4,5)P2 domains assist FCHo2 docking at specific membrane regions, where it self-assembles into ring-like-shaped protein patches. We show that the binding of FCHo2 on cellular membranes promotes PI(4,5)P2 clustering at the boundary of cargo receptors and that this accumulation enhances clathrin assembly. Thus, our results provide a mechanistic framework that could explain the recruitment of early PI(4,5)P2-interacting proteins at endocytic sites.

Structural Basis of Transcription: RNA Polymerase Backtracking and Its Reactivation.

Regulatory sequences or erroneous incorporations during DNA transcription cause RNA polymerase backtracking and inactivation in all kingdoms of life. Reactivation requires RNA transcript cleavage. Essential transcription factors (GreA and GreB, or TFIIS) accelerate this reaction. We report four cryo-EM reconstructions of Escherichia coli RNA polymerase representing the entire reaction pathway: (1) a backtracked complex; a backtracked complex with GreB (2) before and (3) after RNA cleavage; and (4) a reactivated, substrate-bound complex with GreB before RNA extension. Compared with eukaryotes, the backtracked RNA adopts a different conformation. RNA polymerase conformational changes cause distinct GreB states: a fully engaged GreB before cleavage; a disengaged GreB after cleavage; and a dislodged, loosely bound GreB removed from the active site to allow RNA extension. These reconstructions provide insight into the catalytic mechanism and dynamics of RNA cleavage and extension and suggest how GreB targets backtracked complexes without interfering with canonical transcription.

Crystal structure of the Rad3/XPD regulatory domain of Ssl1/p44.

The Ssl1/p44 subunit is a core component of the yeast/mammalian general transcription factor TFIIH, which is involved in transcription and DNA repair. Ssl1/p44 binds to and stimulates the Rad3/XPD helicase activity of TFIIH. To understand the helicase stimulatory mechanism of Ssl1/p44, we determined the crystal structure of the N-terminal regulatory domain of Ssl1 from Saccharomyces cerevisiae. Ssl1 forms a von Willebrand factor A fold in which a central six-stranded ß-sheet is sandwiched between three α helices on both sides. Structural and biochemical analyses of Ssl1/p44 revealed that the ß4-α5 loop, which is frequently found at the interface between von Willebrand factor A family proteins and cellular counterparts, is critical for the stimulation of Rad3/XPD. Yeast genetics analyses showed that double mutation of Leu-239 and Ser-240 in the ß4-α5 loop of Ssl1 leads to lethality of a yeast strain, demonstrating the importance of the Rad3-Ssl1 interactions to cell viability. Here, we provide a structural model for the Rad3/XPD-Ssl1/p44 complex and insights into how the binding of Ssl1/p44 contributes to the helicase activity of Rad3/XPD and cell viability.

Construction of a reporter vector for analysis of bidirectional transcriptional activity of retrovirus LTR.

To study the transcriptional activity of the HIV-1 LTR, we constructed a vector containing Renilla and Firefly luciferase genes under the control of the LTR (wild-type or mutated version) and oriented in a manner that allowed them to be transcribed in opposite directions. We found that the HIV-1 LTR acted as a bidirectional promoter, which activity was controlled by NF-κB- and Sp1-binding sites in both orientations. We next analyzed with this reporter vector the bidirectional promoter activity of the HTLV-1 LTR and showed that this LTR also possessed a bidirectional transcriptional activity. Interestingly, Sp1-binding elements were also involved in the control of HTLV-1 bidirectional transcription. Moreover, both retroviral trans-activators, Tat and Tax, could preferentially activate sense transcription with no or limited effect on the extent of antisense transcription. We also cloned into this plasmid the MLV LTR and found that the LTR of a simple retrovirus also possessed bidirectional transcriptional activity. This reporter vector represents a powerful tool to analyze the bidirectional transcriptional activity of retrovirus LTRs.

HIV-1 antisense transcription is preferentially activated in primary monocyte-derived cells.

In this study, an antisense luciferase-expressing human immunodeficiency virus type 1 (HIV-1) molecular clone was used to infect primary cells. We found that antisense transcription activity from the 3' long terminal repeat (LTR) was significantly more abundant in monocyte-derived cells than in activated T lymphocytes. Moreover, by analyzing antisense transcription in infected monocyte-derived dendritic cells (MDDCs), we observed that the majority of HIV-1-infected MDDCs with significant antisense transcription activity did not produce Gag. We also confirmed that the negative-strand-encoded antisense protein (ASP) was expressed in monocyte-derived cells.

Polarized expression of the membrane ASP protein derived from HIV-1 antisense transcription in T cells.

BACKGROUND: Retroviral gene expression generally depends on a full-length transcript that initiates in the 5' LTR, which is either left unspliced or alternatively spliced. We and others have demonstrated the existence of antisense transcription initiating in the 3' LTR in human lymphotropic retroviruses, including HTLV-1, HTLV-2, and HIV-1. Such transcripts have been postulated to encode antisense proteins important for the establishment of viral infections. The antisense strand of the HIV-1 proviral DNA contains an ORF termed asp, coding for a highly hydrophobic protein. However, although anti-ASP antibodies have been described to be present in HIV-1-infected patients, its in vivo expression requires further support. The objective of this present study was to clearly demonstrate that ASP is effectively expressed in infected T cells and to provide a better characterization of its subcellular localization. RESULTS: We first investigated the subcellular localization of ASP by transfecting Jurkat T cells with vectors expressing ASP tagged with the Flag epitope to its N-terminus. Using immunofluorescence microscopy, we found that ASP localized to the plasma membrane in transfected Jurkat T cells, but with different staining patterns. In addition to an entire distribution to the plasma membrane, ASP showed an asymmetric localization and could also be detected in membrane connections between two cells. We then infected Jurkat T cells with NL4.3 virus coding for ASP tagged with the Flag epitope at its C-terminal end. By this approach, we were capable of showing that ASP is effectively expressed from the HIV-1 3' LTR in infected T cells, with an asymmetric localization of the viral protein at the plasma membrane. CONCLUSION: These results demonstrate for the first time that ASP can be detected when expressed from full-length HIV-1 proviral DNA and that its localization is consistent with Jurkat T cells overexpressing ASP.

The PDZ domain-binding motif of the human T cell leukemia virus type 1 tax protein induces mislocalization of the tumor suppressor hScrib in T cells.

Interactions with cellular PDZ domain-containing proteins obviously contribute to the tumorigenic potential of several viral oncoproteins. In this regard, the oncogenic potential of the human T cell leukemia virus type 1 Tax protein correlates with its binding capacity to the tumor suppressor hDlg. Recent results show that hDlg in T cells is associated to a network of scaffolding proteins including another PDZ domain-containing protein termed hScrib. Interestingly, previous studies have revealed complementary activities of both proteins in the control of epithelial cell polarity. Here, we demonstrate that Tax can bind to hScrib and that the resulting Tax/hScrib complex is present in human T cell leukemia virus type 1-infected T cells. By confocal microscopy, we show that Tax modifies the localization of hScrib in transfected COS cells as well as in infected T cell lines and targets hScrib to particular spots exhibiting a granular distribution, mainly distributed in the cytoplasm. Given that Tax sequesters hScrib to these particular structures, we postulate that Tax might inhibit hScrib activity. Providing further support to this idea, we find that transient overexpression of hScrib attenuates T cell receptor-induced NFAT activity but that the presence of Tax counteracts this negative effect on the NFAT pathway. The fact that hDlg and hScrib are both targeted by Tax underlies their importance in T cell function.

The HBZ-SP1 isoform of human T-cell leukemia virus type I represses JunB activity by sequestration into nuclear bodies.

BACKGROUND: The human T-cell leukemia virus type I (HTLV-I) basic leucine-zipper factor (HBZ) has previously been shown to modulate transcriptional activity of Jun family members. The presence of a novel isoform of HBZ, termed HBZ-SP1, has recently been characterized in adult T-cell leukemia (ATL) cells and has been found to be associated with intense nuclear spots. In this study, we investigated the role of these nuclear bodies in the regulation of the transcriptional activity of JunB. RESULTS: Using fluorescence microscopy, we found that the HBZ-SP1 protein localizes to intense dots corresponding to HBZ-NBs and to nucleoli. We analyzed the relative mobility of the EGFP-HBZ-SP1 fusion protein using fluorescence recovery after photobleaching (FRAP) analysis and found that the deletion of the ZIP domain perturbs the association of the HBZ-SP1 protein to the HBZ-NBs. These data suggested that HBZ needs cellular partners, including bZIP factors, to form HBZ-NBs. Indeed, by cotransfection experiments in COS cells, we have found that the bZIP factor JunB is able to target delocalized form of HBZ (deleted in its nuclear localization subdomains) into the HBZ-NBs. We also show that the viral protein is able to entail a redistribution of JunB into the HBZ-NBs. Moreover, by transfecting HeLa cells (known to express high level of JunB) with a vector expressing HBZ-SP1, the sequestration of JunB to the HBZ-NBs inhibited its transcriptional activity. Lastly, we analyzed the nuclear distribution of HBZ-SP1 in the presence of JunD, a Jun family member known to be activated by HBZ. In this case, no NBs were detected and the HBZ-SP1 protein was diffusely distributed throughout the nucleoplasm. CONCLUSION: Our results suggest that HBZ-mediated sequestration of JunB to the HBZ-NBs may be causing the repression of JunB activity in vivo.

A modified version of a Fos-associated cluster in HBZ affects Jun transcriptional potency.

Like c-Fos, HBZ (HTLV-I bZIP factor) is able to interact with c-Jun but differs considerably from c-Fos in its ability to activate AP-1-responsive genes since HBZ rather inhibits transcriptional activity of c-Jun. To better understand the molecular mechanisms involved in this down-regulation of c-Jun activity, a large number of HBZ/c-Fos chimeras was constructed and analyzed for their ability to interact with c-Jun, to bind to the AP-1 motif and to stimulate expression of a reporter gene containing the collagenase promoter. By this approach, we demonstrate that the DNA-binding domain of HBZ is responsible for its inhibitory effect on the trans-activation potential of c-Jun. However, unexpectedly, we found that exchange of a cluster of six charged amino acids immediately adjacent to the DNA contact region altered significantly transcriptional activity of chimeras. This particular subdomain could be involved in efficient presentation of the AP-1 complex to the transcriptional machinery. To confirm this role, specific residues present in the cluster of HBZ were substituted for corresponding amino acids in c-Fos. Unlike the JunD-activating potential of wild-type HBZ, this mutant was no longer able to stimulate JunD activity, confirming the key role of this particular cluster in regulation of Jun transcriptional potency.

HTLV-I antisense transcripts initiating in the 3'LTR are alternatively spliced and polyadenylated.

BACKGROUND: Antisense transcription in retroviruses has been suggested for both HIV-1 and HTLV-I, although the existence and coding potential of these transcripts remain controversial. Thorough characterization is required to demonstrate the existence of these transcripts and gain insight into their role in retrovirus biology. RESULTS: This report provides the first complete characterization of an antisense retroviral transcript that encodes the previously described HTLV-I HBZ protein. In this study, we show that HBZ-encoding transcripts initiate in the 3' long terminal repeat (LTR) at several positions and consist of two alternatively spliced variants (SP1 and SP2). Expression of the most abundant HBZ spliced variant (SP1) could be detected in different HTLV-I-infected cell lines and importantly in cellular clones isolated from HTLV-I-infected patients. Polyadenylation of HBZ RNA occurred at a distance of 1450 nucleotides downstream of the HBZ stop codon in close proximity of a typical polyA signal. We have also determined that translation mostly initiates from the first exon located in the 3' LTR and that the HBZ isoform produced from the SP1 spliced variant demonstrated inhibition of Tax and c-Jun-dependent transcriptional activation. CONCLUSION: These results conclusively demonstrate the existence of antisense transcription in retroviruses, which likely plays a role in HTLV-I-associated pathogenesis through HBZ protein synthesis.

Nuclear localization of HTLV-I bZIP factor (HBZ) is mediated by three distinct motifs.

The genome of the human T-cell leukemia virus type I (HTLV-I) codes for a basic leucine zipper protein, HBZ, capable of repressing JUN activity and viral transcription. Transient expression in mammalian cells showed that HBZ was targeted to the nucleus, where it accumulated in nuclear speckles. By using a complementary set of deletion mutants, we report here that the nuclear targeting of HBZ is mediated by three distinct nuclear localization signals and that at least two are necessary for the translocation of HBZ to the nucleus. Moreover, the resulting mutant proteins distribute throughout the nucleoplasm and/or into the nucleoli, whereas the wild-type HBZ exclusively accumulates in nuclear speckles, suggesting that the integrity of the protein is required for its speckle localization. We also demonstrate that the HBZ-containing speckles do not correspond to Cajal bodies, splicing factor compartments, or promyelocytic leukemia oncoprotein bodies. Unexpectedly, by using immunogold electron microscopy, we found HBZ localized to heterochromatin. Until now, such characteristics had never been described for a transcription factor and could explain the inhibitory activity of HBZ.