Christiane Branlant

2-D Structure of the A Region of Xist RNA and Its Implication for PRC2 Association

Structural analyses provide new insights into the folding of the A region of the Xist RNA, which plays a crucial role in X chromosome inactivation, and its mechanism of protein recruitment.

The Hsp90 chaperone controls the biogenesis of L7Ae RNPs through conserved machinery

RNA-binding proteins of the L7Ae family are at the heart of many essential ribonucleoproteins (RNPs), including box C/D and H/ACA small nucleolar RNPs, U4 small nuclear RNP, telomerase, and messenger RNPs coding for selenoproteins. In this study, we show that Nufip and its yeast homologue Rsa1 are key components of the machinery that assembles these RNPs. We observed that Rsa1 and Nufip bind several L7Ae proteins and tether them to other core proteins in the immature particles. Surprisingly, Rsa1 and Nufip also link assembling RNPs with the AAA + adenosine triphosphatases hRvb1 and hRvb2 and with the Hsp90 chaperone through two conserved adaptors, Tah1/hSpagh and Pih1. Inhibition of Hsp90 in human cells prevents the accumulation of U3, U4, and telomerase RNAs and decreases the levels of newly synthesized hNop58, hNHP2, 15.5K, and SBP2. Thus, Hsp90 may control the folding of these proteins during the formation of new RNPs. This suggests that Hsp90 functions as a master regulator of cell proliferation by allowing simultaneous control of cell signaling and cell growth.

Pseudouridine Mapping in the Saccharomyces cerevisiae Spliceosomal U Small Nuclear RNAs (snRNAs) Reveals that Pseudouridine Synthase Pus1p Exhibits a Dual Substrate Specificity for U2 snRNA and tRNA

ABSTRACT Pseudouridine (Ψ) residues were localized in theSaccharomyces cerevisiae spliceosomal U small nuclear RNAs (UsnRNAs) by using the chemical mapping method. In contrast to vertebrate UsnRNAs, S. cerevisiae UsnRNAs contain only a few Ψ residues, which are located in segments involved in intermolecular RNA-RNA or RNA-protein interactions. At these positions, UsnRNAs are universally modified. When yeast mutants disrupted for one of the several pseudouridine synthase genes (PUS1,PUS2, PUS3, and PUS4) or depleted in rRNA-pseudouridine synthase Cbf5p were tested for UsnRNA Ψ content, only the loss of the Pus1p activity was found to affect Ψ formation in spliceosomal UsnRNAs. Indeed, Ψ44 formation in U2 snRNA was abolished. By using purified Pus1p enzyme and in vitro-produced U2 snRNA, Pus1p is shown here to catalyze Ψ44 formation in the S. cerevisiae U2 snRNA. Thus, Pus1p is the first UsnRNA pseudouridine synthase characterized so far which exhibits a dual substrate specificity, acting on both tRNAs and U2 snRNA. As depletion of rRNA-pseudouridine synthase Cbf5p had no effect on UsnRNA Ψ content, formation of Ψ residues in S. cerevisiae UsnRNAs is not dependent on the Cbf5p-snoRNA guided mechanism.

A Janus Splicing Regulatory Element Modulates HIV-1 tat and rev mRNA Production by Coordination of hnRNP A1 Cooperative Binding

Retroviral protein production depends upon alternative splicing of the viral transcript. The HIV-1 acceptor site A7 is required for tat and rev mRNA production. Production of the Tat transcriptional activator is highly controlled because of its apoptotic properties. Two silencer elements (ESS3 and ISS) and two enhancer elements (ESE2 and ESE3/(GAA)3) were previously identified at site A7. hnRNP A1 binds ISS and ESS3 and is involved in the inhibitory process, ASF/SF2 activates site A7 utilisation. Here, by using chemical and enzymatic probes we established the 2D structure of the HIV-1(BRU) RNA region containing site A7 and identified the RNA segments protected in nuclear extract and by purified hnRNP A1. ISS, ESE3/(GAA)3 and ESS3 are located in three distinct stem-loop structures (SLS1, 2 and 3). As expected, hnRNP A1 binds sites 1, 2 and 3 of ISS and ESS3b, and oligomerises on the polypurine sequence upstream of ESS3b. In addition, we discovered an unidentified hnRNP A1 binding site (AUAGAA), that overlaps ESE3/(GAA)3. On the basis of competition experiments, hnRNP A1 has a stronger affinity for this site than for ESS3b. By insertion of (GAA)3 alone or preceded by the AUA trinucleotide in a foreign context, the AUAGAA sequence was found to modulate strongly the (GAA)3 splicing enhancer activity. Cross-linking experiments on these heterologous RNAs and the SLS2-SLS3 HIV-1 RNA region, in nuclear extract and with recombinant proteins, showed that binding of hnRNP A1 to AUA(GAA)3 strongly competes the association of ASF/SF2 with (GAA)3. In addition, disruption of AUA(GAA)3 demonstrated a key role of this sequence in hnRNP A1 cooperative binding to the ISS and ESS3b inhibitors and hnRNP A1 oligomerisation on the polypurine sequence. Thus, depending on the cellular context ([ASF/SF2]/[hnRNP A1] ratio), AUA(GAA)3 will activate or repress site A7 utilisation and can thus be considered as a Janus splicing regulator.

In Vitro and in Cellulo Evidences for Association of the Survival of Motor Neuron Complex with the Fragile X Mental Retardation Protein

Spinal muscular atrophy (SMA) is caused by reduced levels of the survival of motor neuron (SMN) protein. Although the SMN complex is essential for assembly of spliceosomal U small nuclear RNPs, it is still not understood why reduced levels of the SMN protein specifically cause motor neuron degeneration. SMN was recently proposed to have specific functions in mRNA transport and translation regulation in neuronal processes. The defective protein in Fragile X mental retardation syndrome (FMRP) also plays a role in transport of mRNPs and in their translation. Therefore, we examined possible relationships of SMN with FMRP. We observed granules containing both transiently expressed red fluorescent protein(RFP)-tagged SMN and green fluorescent protein(GFP)-tagged FMRP in cell bodies and processes of rat primary neurons of hypothalamus in culture. By immunoprecipitation experiments, we detected an association of FMRP with the SMN complex in human neuroblastoma SH-SY5Y cells and in murine motor neuron MN-1 cells. Then, by in vitro experiments, we demonstrated that the SMN protein is essential for this association. We showed that the COOH-terminal region of FMRP, as well as the conserved YG box and the region encoded by exon 7 of SMN, are required for the interaction. Our findings suggest a link between the SMN complex and FMRP in neuronal cells.

Small-Molecule Inhibition of HIV pre-mRNA Splicing as a Novel Antiretroviral Therapy to Overcome Drug Resistance

The development of multidrug-resistant viruses compromises antiretroviral therapy efficacy and limits therapeutic options. Therefore, it is an ongoing task to identify new targets for antiretroviral therapy and to develop new drugs. Here, we show that an indole derivative (IDC16) that interferes with exonic splicing enhancer activity of the SR protein splicing factor SF2/ASF suppresses the production of key viral proteins, thereby compromising subsequent synthesis of full-length HIV-1 pre-mRNA and assembly of infectious particles. IDC16 inhibits replication of macrophage- and T cell–tropic laboratory strains, clinical isolates, and strains with high-level resistance to inhibitors of viral protease and reverse transcriptase. Importantly, drug treatment of primary blood cells did not alter splicing profiles of endogenous genes involved in cell cycle transition and apoptosis. Thus, human splicing factors represent novel and promising drug targets for the development of antiretroviral therapies, particularly for the inhibition of multidrug-resistant viruses.

Alternative splicing: regulation of HIV-1 multiplication as a target for therapeutic action

The retroviral life cycle requires that significant amounts of RNA remain unspliced and perform several functions in the cytoplasm. Thus, the full‐length RNA serves both the viral genetic material that will be encapsulated in viral particles and the mRNA encoding structural and enzymatic proteins required for viral replication. Simple retroviruses produce one single‐spliced env RNA from the full‐length precursor RNA, whereas complex retroviruses, such as HIV, are characterized by the production of multiple‐spliced RNA species. In this review we will summarize the current acknowledge about the HIV‐1 alternative splicing mechanism and will describe how this malleable process can help further understanding of infection, spread and dissemination through splicing regulation. Such studies coupled with the testing of splicing inhibitors should help the development of new therapeutic antiviral agents.

Conserved loop I of U5 small nuclear RNA is dispensable for both catalytic steps of pre-mRNA splicing in HeLa nuclear extracts.

ABSTRACT The function of conserved regions of the metazoan U5 snRNA was investigated by reconstituting U5 small nuclear ribonucleoprotein particles (snRNPs) from purified snRNP proteins and HeLa orXenopus U5 snRNA mutants and testing their ability to restore splicing to U5-depleted nuclear extracts. Substitution of conserved nucleotides comprising internal loop 2 or deletion of internal loop 1 had no significant effect on the ability of reconstituted U5 snRNPs to complement splicing. However, deletion of internal loop 2 abolished U5 activity in splicing and spliceosome formation. Surprisingly, substitution of the invariant loop 1 nucleotides with a GAGA tetraloop had no effect on U5 activity. Furthermore, U5 snRNPs reconstituted from an RNA formed by annealing the 5′ and 3′ halves of the U5 snRNA, which lacked all loop 1 nucleotides, complemented both steps of splicing. Thus, in contrast to yeast, loop 1 of the human U5 snRNA is dispensable for both steps of splicing in HeLa nuclear extracts. This suggests that its function can be compensated for in vitro by other spliceosomal components: for example, by proteins associated with the U5 snRNP. Consistent with this idea, immunoprecipitation studies indicated that several functionally important U5 proteins associate stably with U5 snRNPs containing a GAGA loop 1 substitution.

Biochemical and NMR Study on the Competition between Proteins SC35, SRp40, and Heterogeneous Nuclear Ribonucleoprotein A1 at the HIV-1 Tat Exon 2 Splicing Site

The human immunodeficiency virus, type 1, Tat protein plays a key role in virus multiplication. Because of its apoptotic property, its production is highly controlled. It depends upon the A3 splicing site utilization. A key control of site A3 activity is the ESS2 splicing silencer, which is located within the long stem-loop structure 3 (SLS3), far downstream from site A3. Here, by enzymatic footprints, we demonstrate the presence of several heterogeneous nuclear ribonucleoprotein (hnRNP) A1-binding sites on SLS3 and show the importance of the C-terminal Gly domain of hnRNP A1 in the formation of stable complexes containing several hnRNP A1 molecules bound on SLS3. Mutations in each of the UAG triplets in ESS2 strongly reduce the overall hnRNP A1 binding, showing the central role of ESS2 in hnRNP A1 assembly on SLS2-SLS3. Using NMR spectroscopy, we demonstrate the direct interaction of ESS2 with the RNA recognition motifs domains of hnRNP A1. This interaction has limited effect on the RNA two-dimensional structure. The SR proteins SC35 and SRp40 were found previously to be strong activators of site A3 utilization. By enzymatic and chemical footprints, we delineate their respective binding sites on SLS2 and SLS3 and find a strong similarity between the hnRNP A1-, SC35-, and SRp40-binding sites. The strongest SC35-binding site only has a modest contribution to site A3 activation. Hence, the main role of SR proteins at site A3 is to counteract hnRNP A1 binding on ESS2 and ESE2. Indeed, we found that ESE2 has inhibitory properties because of its ability to bind hnRNP A1.

The strong efficiency of the Escherichia coli gapA P1 promoter depends on a complex combination of functional determinants

The Escherichia coli multi-promoter region of the gapA gene ensures a high level of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) production under various growth conditions. In the exponential phase of growth, gapA mRNAs are mainly initiated at the highly efficient gapA P1 promoter. In the present study, by using site-directed mutagenesis and chemical probing of the RPo (open complex) formed by Esigma70 (holoenzyme associated with sigma70) RNAP (RNA polymerase) at promoter gapA P1, we show that this promoter is an extended -10 promoter that needs a -35 sequence for activity. The -35 sequence compensates for the presence of a suboptimal -10 hexamer. A tract of thymine residues in the spacer region, which is responsible for a DNA distortion, is also required for efficient activity. We present the first chemical probing of an RPo formed at a promoter needing both a -10 extension and a -35 sequence. It reveals a complex array of RNAP-DNA interactions. In agreement with the fact that residue A-11 in the non-template strand is flipped out in a protein pocket in previously studied RPos, the corresponding A residue in gapA P1 promoter is protected in RPo and is essential for activity. However, in contrast with some of the previous findings on RPos formed at other promoters, the -12 A:T pair is opened. Strong contacts with RNAP occur both with the -35 sequence and the TG extension, so that the sigma4 and sigma2 domains may simultaneously contact the promoter DNA. RNAP-DNA interactions were also detected immediately downstream of the -35 hexamer and in a more distal upstream segment, reflecting a wrapping of RNAP by the core and upstream promoter DNA. Altogether, the data reveal that promoter gapA P1 is a very efficient promoter sharing common properties with both extended -10 and non-extended -10 promoters.