Iván D'Orso

Host Cell Factors in HIV Replication: Meta-Analysis of Genome-Wide Studies

We have analyzed host cell genes linked to HIV replication that were identified in nine genome-wide studies, including three independent siRNA screens. Overlaps among the siRNA screens were very modest (<7% for any pairwise combination), and similarly, only modest overlaps were seen in pairwise comparisons with other types of genome-wide studies. Combining all genes from the genome-wide studies together with genes reported in the literature to affect HIV yields 2,410 protein-coding genes, or fully 9.5% of all human genes (though of course some of these are false positive calls). Here we report an “encyclopedia” of all overlaps between studies (available at http://www.hostpathogen.org), which yielded a more extensively corroborated set of host factors assisting HIV replication. We used these genes to calculate refined networks that specify cellular subsystems recruited by HIV to assist in replication, and present additional analysis specifying host cell genes that are attractive as potential therapeutic targets.

RNA-mediated displacement of an inhibitory snRNP complex activates transcription elongation

The transition from transcription initiation to elongation at the HIV-1 promoter is controlled by Tat, which recruits P-TEFb to TAR RNA to phosphorylate RNA polymerase II. It has long been unclear why the HIV-1 promoter is incompetent for elongation. We report that P-TEFb is recruited to the promoter in a catalytically inactive state bound to the inhibitory 7SK small nuclear ribonucleoprotein (snRNP), thereby preventing elongation. It also has long been believed that TAR functions to recruit Tat to the promoter, but we find that Tat is recruited to the DNA template before TAR is synthesized. We propose that TAR binds Tat and P-TEFb as it emerges on the nascent transcript, competitively displacing the inhibitory 7SK snRNP and activating the P-TEFb kinase. Recruitment of an inhibitory snRNP complex at an early stage in the transcription cycle provides a new paradigm for controlling gene expression with a noncoding RNA.

A solution to limited genomic capacity: using adaptable binding surfaces to assemble the functional HIV Rev oligomer on RNA

Many ribonucleoprotein (RNP) complexes assemble into large, organized structures in which protein subunits are positioned by interactions with RNA and other proteins. Here we demonstrate that HIV Rev, constrained in size by a limited viral genome, also forms an organized RNP by assembling a homo-oligomer on the Rev response element (RRE) RNA. Rev subunits bind cooperatively to discrete RNA sites using an oligomerization domain and an adaptable protein-RNA interface, forming a complex with 500-fold higher affinity than the tightest single interaction. High-affinity binding correlates strongly with RNA export activity. Rev utilizes different surfaces of its alpha-helical RNA-binding domain to recognize several low-affinity binding sites, including the well-characterized stem IIB site and an additional site in stem IA. We propose that adaptable RNA-binding surfaces allow the Rev oligomer to assemble economically into a discrete, stable RNP and provide a mechanistic role for Rev oligomerization during the HIV life cycle.

The Trypanosoma cruzi Mucin Family Is Transcribed from Hundreds of Genes Having Hypervariable Regions

In previous works we have identified genes in the protozoan parasite Trypanosoma cruzi whose structure resemble those of mammalian mucin genes. Indirect evidence suggested that these genes might encode the core protein of parasite mucins, glycoproteins that were proposed to be involved in the interaction with, and invasion of, mammalian host cells. We now show that the mucin gene family from T. cruzi is much larger and diverse than expected. A minimal number of 484 mucin genes per haploid genome is calculated for a parasite clone. Most, if not all, genes are transcribed, as deduced from cDNA analysis. Comparison of the cDNA sequences showed evidences of a high mutation rate in localized regions of the genes. Sequence conservation among members of the family is much higher in the untranslated (UTR) regions than in the sequences encoding the mature mucin core protein. Transcription units can be classified into two main subfamilies according to the sequence homologies in the 5′-UTR, whereas the 3′-UTR is highly conserved in all clones analyzed. The common origin of members of this gene family as well as their relationships can be defined by sequence comparison of different domains in the transcription units. The regions encoding the N and C termini, supposed to correspond to the leader peptide and membrane-anchoring signal, respectively, (Di Noia, J. M., Sánchez, D. O., and Frasch, A. C. C. (1995)J. Biol. Chem. 270, 24146–24149) are highly conserved. Conversely, the central regions are highly variable. These regions encode the target sites for O-glycosylation and are made of a variable number of repetitive units rich in Thr and Pro residues or are nonrepetitive but still rich in Thr/Ser and Pro residues. The region putatively coding for the N-terminal domain of the mature core protein is hypervariable, being different in most of the transcripts sequenced. Nonrepetitive central domains are unique to each gene. Gene-specific probes show that the relative abundance of different mRNAs varies greatly within the same parasite clone.

Tat acetylation modulates assembly of a viral-host RNA–protein transcription complex

HIV-1 Tat enhances viral transcription elongation by forming a ribonucleoprotein complex with transactivating responsive (TAR) RNA and P-TEFb, an elongation factor composed of cyclin T1 (CycT1) and Cdk9 that phosphorylates the C-terminal domain of RNA polymerase II. Previous studies have shown that Lys-28 in the activation domain (AD) of Tat is essential for HIV-1 transcription and replication and is acetylated by p300/CBP-associated factor (PCAF), but the mechanistic basis of the Lys-28 requirement is unknown. Here, we show that Lys-28 acetylation modulates the affinity and stability of HIV-1 Tat–CycT1–TAR complexes by enhancing an interaction with the CycT1 Tat–TAR recognition motif. High-affinity assembly correlates strongly with stimulation of transcription elongation in vitro and Tat activation in vivo. In marked contrast, bovine lentiviral Tat proteins have evolved a high-affinity TAR interaction that does not require PCAF-mediated acetylation of the Tat AD or CycT1 for RNA binding, whereas HIV-2 Tat has evolved an intermediate mechanism that uses a duplicated TAR element and CycT1 to enhance RNA affinity and consequently transcription activation. The coevolution of Tat acetylation, CycT1 dependence, and TAR binding affinity is seen in viral replication assays using Tat proteins that rely on CycT1 for TAR binding but are acetylation deficient, where compensatory mutations rapidly accrue in TAR to generate high-affinity, CycT1-independent complexes reminiscent of the bovine viruses. Thus, lysine acetylation can be used to modulate and evolve the strength of a viral-host RNA–protein complex, thereby tuning the levels of transcription elongation.

mRNA maturation by two-step trans-splicing/polyadenylation processing in trypanosomes

Trypanosomes are unique eukaryotic cells, in that they virtually lack mechanisms to control gene expression at the transcriptional level. These microorganisms mostly control protein synthesis by posttranscriptional regulation processes, like mRNA stabilization and degradation. Transcription in these cells is polycistronic. Tens to hundreds of protein-coding genes of unrelated function are arrayed in long clusters on the same DNA strand. Polycistrons are cotranscriptionally processed by trans-splicing at the 5′ end and polyadenylation at the 3′ end, generating monocistronic units ready for degradation or translation. In this work, we show that some trans-splicing/polyadenylation sites may be skipped during normal polycistronic processing. As a consequence, dicistronic units or monocistronic transcripts having long 3′ UTRs are produced. Interestingly, these unspliced transcripts can be processed into mature mRNAs by the conventional trans-splicing/polyadenylation events leading to translation. To our knowledge, this is a previously undescribed mRNA maturation by trans-splicing uncoupled from transcription. We identified an RNA-recognition motif-type protein, homologous to the mammalian polypyrimidine tract-binding protein, interacting with one of the partially processed RNAs analyzed here that might be involved in exon skipping. We propose that splice-site skipping might be part of a posttranscriptional mechanism to regulate gene expression in trypanosomes, through the generation of premature nontranslatable RNA molecules.

Small trypanosome RNA-binding proteins TbUBP1 and TbUBP2 influence expression of F-box protein mRNAs in bloodstream trypanosomes.

ABSTRACT In the African trypanosome Trypanosoma brucei nearly all control of gene expression is posttranscriptional; sequences in the 3′-untranslated regions of mRNAs determine the steady-state mRNA levels by regulation of RNA turnover. Here we investigate the roles of two related proteins, TbUBP1 and TbUBP2, containing a single RNA recognition motif, in trypanosome gene expression. TbUBP1 and TbUBP2 are in the cytoplasm and nucleus, comprise ca. 0.1% of the total protein, and are not associated with polysomes or RNA degradation enzymes. Overexpression of TbUBP2 upregulated the levels of several mRNAs potentially involved in cell division, including the CFB1 mRNA, which encodes a protein with a cyclin F-box domain. CFB1 regulation was mediated by the 3′-untranslated region and involved stabilization of the mRNA. Depletion of TbUBP2 and TbUBP1 inhibited growth and downregulated expression of the cyclin F box protein gene CFB2; trans splicing was unaffected. The results of pull-down assays indicated that all tested mRNAs were bound to TbUBP2 or TbUBP1, with some preference for CFB1. We suggest that TbUBP1 and TbUBP2 may be relatively nonspecific RNA-binding proteins and that specific effects of overexpression or depletion could depend on competition between various different proteins for RNA binding.

Transition Step during Assembly of HIV Tat:P-TEFb Transcription Complexes and Transfer to TAR RNA

ABSTRACT Transcription factors regulate eukaryotic RNA polymerase II (Pol II) activity by assembling and remodeling complexes at multiple steps in the transcription cycle. In HIV, we previously proposed a two-step model where the viral Tat protein first preassembles at the promoter with an inactive P-TEFb:7SK snRNP complex and later transfers P-TEFb to TAR on the nascent transcript, displacing the inhibitory snRNP and resulting in Pol II phosphorylation and stimulation of elongation. It is unknown how the Tat:P-TEFb complex transitions to TAR to activate the P-TEFb kinase. Here, we show that P-TEFb artificially recruited to the nascent transcript is not competent for transcription but rather remains inactive due to its assembly with the 7SK snRNP. Tat supplied in trans is able to displace the kinase inhibitor Hexim1 from the snRNP and activate P-TEFb, thereby uncoupling Tat requirements for kinase activation and TAR binding. By combining comprehensive mutagenesis of Tat with multiple cell-based reporter assays that probe the activity of Tat in different arrangements, we genetically defined a transition step in which preassembled Tat:P-TEFb complexes switch to TAR. We propose that a conserved network of residues in Tat has evolved to control this transition and thereby switch the host elongation machinery to viral transcription.

PPM1G Binds 7SK RNA and Hexim1 To Block P-TEFb Assembly into the 7SK snRNP and Sustain Transcription Elongation

ABSTRACT Transcription elongation programs are vital for the precise regulation of several biological processes. One key regulator of such programs is the P-TEFb kinase, which phosphorylates RNA polymerase II (Pol II) once released from the inhibitory 7SK small nuclear ribonucleoprotein (snRNP) complex. Although mechanisms of P-TEFb release from the snRNP are becoming clearer, how P-TEFb remains in the 7SK-unbound state to sustain transcription elongation programs remains unknown. Here we report that the PPM1G phosphatase (inducibly recruited by nuclear factor κB [NF-κB] to target promoters) directly binds 7SK RNA and the kinase inhibitor Hexim1 once P-TEFb has been released from the 7SK snRNP. This dual binding activity of PPM1G blocks P-TEFb reassembly onto the snRNP to sustain NF-κB-mediated Pol II transcription in response to DNA damage. Notably, the PPM1G-7SK RNA interaction is direct, kinetically follows the recruitment of PPM1G to promoters to activate NF-κB transcription, and is reversible, since the complex disassembles before resolution of the program. Strikingly, we found that the ataxia telangiectasia mutated (ATM) kinase regulates the interaction between PPM1G and the 7SK snRNP through site-specific PPM1G phosphorylation. The precise and temporally regulated interaction of a cellular enzyme and a noncoding RNA provides a new paradigm for simultaneously controlling the activation and maintenance of inducible transcription elongation programs.

Transcription control by long non-coding RNAs

Non-coding RNAs have been found to regulate many cellular processes and thus expand the functional genetic repertoire contained within the genome. With the recent advent of genomic tools, it is now evident that these RNA molecules play central regulatory roles in many transcriptional programs. Here we discuss how they are targeted to promoters in several cases and how they operate at specific points in the transcription cycle to precisely control gene expression.