Fused in Liposarcoma Protein, a New Player in the Regulation of HIV-1 Transcription, Binds to Known and Newly Identified LTR G-Quadruplexes.

HIV-1 integrated long terminal repeat (LTR) promoter activity is modulated by folding of its G-rich region into non-canonical nucleic acids structures, such as G-quadruplexes (G4s), and their interaction with cellular proteins. Here, by a combined pull-down/mass spectrometry/Western-blot approach, we identified the fused in liposarcoma (FUS) protein and found it to preferentially bind and stabilize the least stable and bulged LTR G4, especially in the cell environment. The outcome of this interaction is the down-regulation of viral transcription, as assessed in a reporter assay with LTR G4 mutants in FUS-silencing conditions. These data indicate that the complexity and dynamics of HIV-1 LTR G4s are much greater than previously envisaged. The G-rich LTR region, with its diverse G4 landscape and multiple cell protein interactions, stands out as prime sensing center for the fine regulation of viral transcription. This region thus represents a rational antiviral target for inhibiting both the actively transcribing and latent viruses.

The Roc domain of LRRK2 as a hub for protein-protein interactions: a focus on PAK6 and its impact on RAB phosphorylation.

Leucine-rich repeat kinase 2 (LRRK2) has taken center stage in Parkinson's disease (PD) research as mutations cause familial PD and more common variants increase lifetime risk for disease. One unique feature in LRRK2 is the coexistence of GTPase/Roc (Ras of complex) and kinase catalytic functions, bridged by a COR (C-terminal Of Roc) platform for dimerization. Multiple PD mutations are located within the Roc/GTPase domain and concomitantly lead to defective GTPase activity and augmented kinase activity in cells, supporting a crosstalk between GTPase and kinase domains. In addition, biochemical and structural data highlight the importance of Roc as a molecular switch modulating LRRK2 monomer-to-dimer equilibrium and building the interface for interaction with binding partners. Here we review the effects of PD Roc mutations on LRRK2 function and discuss the importance of Roc as a hub for multiple molecular interactions relevant for the regulation of cytoskeletal dynamics and intracellular trafficking pathways. Among the well-characterized Roc interactors, we focused on the cytoskeletal-related kinase p21-activated kinase 6 (PAK6). We report the affinity between LRRK2-Roc and PAK6 measured by microscale thermophoresis (MST). We further show that PAK6 can modulate LRRK2-mediated phosphorylation of RAB substrates in the presence of LRRK2 wild-type (WT) or the PD G2019S kinase mutant but not when the PD Roc mutation R1441G is expressed. These findings support a mechanism whereby mutations in Roc might affect LRRK2 activity through impaired protein-protein interaction in the cell.

The cellular protein hnRNP A2/B1 enhances HIV-1 transcription by unfolding LTR promoter G-quadruplexes.

G-quadruplexes are four-stranded conformations of nucleic acids that act as cellular epigenetic regulators. A dynamic G-quadruplex forming region in the HIV-1 LTR promoter represses HIV-1 transcription when in the folded conformation. This activity is enhanced by nucleolin, which induces and stabilizes the HIV-1 LTR G-quadruplexes. In this work by a combined pull-down/mass spectrometry approach, we consistently found hnRNP A2/B1 as an additional LTR-G-quadruplex interacting protein. Surface plasmon resonance confirmed G-quadruplex specificity over linear sequences and fluorescence resonance energy transfer analysis indicated that hnRNP A2/B1 is able to efficiently unfold the LTR G-quadruplexes. Evaluation of the thermal stability of the LTR G-quadruplexes in different-length oligonucleotides showed that the protein is fit to be most active in the LTR full-length environment. When hnRNP A2/B1 was silenced in cells, LTR activity decreased, indicating that the protein acts as a HIV-1 transcription activator. Our data highlight a tightly regulated control of transcription based on G-quadruplex folding/unfolding, which depends on interacting cellular proteins. These findings provide a deeper understanding of the viral transcription mechanism and may pave the way to the development of drugs effective against the integrated HIV-1, present both in actively and latently infected cells.

The cellular protein nucleolin preferentially binds long-looped G-quadruplex nucleic acids.

BACKGROUND: G-quadruplexes (G4s) are four-stranded nucleic acid structures that form in G-rich sequences. Nucleolin (NCL) is a cellular protein reported for its functions upon G4 recognition, such as induction of neurodegenerative diseases, tumor and virus mechanisms activation. We here aimed at defining NCL/G4 binding determinants. METHODS: Electrophoresis mobility shift assay was used to detect NCL/G4 binding; circular dichroism to assess G4 folding, topology and stability; dimethylsulfate footprinting to detect G bases involved in G4 folding. RESULTS: The purified full-length human NCL was initially tested on telomeric G4 target sequences to allow for modulation of loop, conformation, length, G-tract number, stability. G4s in promoter regions with more complex sequences were next employed. We found that NCL binding to G4s heavily relies on G4 loop length, independently of the conformation and oligonucleotide/loop sequence. Low stability G4s are preferred. When alternative G4 conformations are possible, those with longer loops are preferred upon binding to NCL, even if G-tracts need to be spared from G4 folding. CONCLUSIONS: Our data provide insight into how G4s and the associated proteins may control the ON/OFF molecular switch to several pathological processes, including neurodegeneration, tumor and virus activation. Understanding these regulatory determinants is the first step towards the development of targeted therapies. GENERAL SIGNIFICANCE: The indication that NCL binding preferentially stimulates and induces folding of G4s containing long loops suggests NCL ability to modify the overall structure and steric hindrance of the involved nucleic acid regions. This protein-induced modification of the G4 structure may represent a cellular mechanosensor mechanism to molecular signaling and disease pathogenesis. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

Nucleolin stabilizes G-quadruplex structures folded by the LTR promoter and silences HIV-1 viral transcription.

Folding of the LTR promoter into dynamic G-quadruplex conformations has been shown to suppress its transcriptional activity in HIV-1. Here we sought to identify the proteins that control the folding of this region of proviral genome by inducing/stabilizing G-quadruplex structures. The implementation of electrophorethic mobility shift assay and pull-down experiments coupled with mass spectrometric analysis revealed that the cellular protein nucleolin is able to specifically recognize G-quadruplex structures present in the LTR promoter. Nucleolin recognized with high affinity and specificity the majority, but not all the possible G-quadruplexes folded by this sequence. In addition, it displayed greater binding preference towards DNA than RNA G-quadruplexes, thus indicating two levels of selectivity based on the sequence and nature of the target. The interaction translated into stabilization of the LTR G-quadruplexes and increased promoter silencing activity; in contrast, disruption of nucleolin binding in cells by both siRNAs and a nucleolin binding aptamer greatly increased LTR promoter activity. These data indicate that nucleolin possesses a specific and regulated activity toward the HIV-1 LTR promoter, which is mediated by G-quadruplexes. These observations provide new essential insights into viral transcription and a possible low mutagenic target for antiretroviral therapy.

General and specific replication profiles are detected in normal human cells by genome-wide and single-locus molecular combing.

Mammalian genomes are replicated under a flexible program, with random use of origins and variable fork rates, and many details of the process must be still unraveled. Molecular combing provides a set of direct data regarding the replication profile of eukaryotic cells: fork rates; organization of the replication clusters; proportion of unidirectional forks; and fork dynamics. In this study the replication profiles of different primary and immortalized non-cancer human cells (lymphocytes, lymphoblastoid cells, fibroblasts) were evaluated at the whole-genome level or within reference genomic regions harboring coding genes. It emerged that these different cell types are characterized by specific replication profiles. In primary fibroblasts, a remarkable fraction of the mammalian genome was found to be replicated by unidirectional forks, and interestingly, the proportion of unidirectional forks further increased in the replicating genome along the population divisions. A second difference concerned in the proportion of paused replication forks, again more frequent in primary fibroblasts than in PBL/lymphoblastoid cells. We concluded that these patterns, whose relevance could escape when genomic methods are applied, represent normal replication features. In single-locus analyses, unidirectional and paused replication forks were highly represented in all genomic regions considered with respect to the average estimates referring to the whole-genome. In addition, fork rates were significantly lower than whole-genome estimates. Instead, when considering the specificities of each genomic region investigated (early to late replication, normal or fragile site) no further differentiating features of replication profiles were detected. These data, representing the integration of genome-wide and single-locus analyses, highlight a large heterogeneity of replication profiles among cell types and within the genome, which should be considered for the correct use of replication datasets.

Genetic instability of the tumor suppressor gene FHIT in normal human cells.

Common fragile sites are hotspots for chromosome instability and co-localize to cancer genomic rearrangements. Whether these loci may be considered stable in human subjects under physiological conditions remains an open question. Here we show by molecular combing that a small but significant percentage of normal human cells carry an abnormal sequence pattern within the tumor suppressor gene FHIT (3p14.2) at FRA3B. Each sequence variation represents a unique pattern within a normal cell population, and therefore it would remain undetected or not interpreted by genome-wide analyses. Remarkably, the region is the same as in FHIT rearrangements described in tumors. By analyses on several normal cell lines (proliferating and resting primary lymphocytes, primary fibroblasts, lymphoblastoid cells including clonal cell cultures) we verified that: (a) each cell type displays altered sequence patterns at FHIT; (b) the presence of abnormal sequence patterns is specific for the FHIT locus; and (c) FHIT instability occurs de novo during cell proliferation, and heterogeneous sequence variants progressively accumulate in the cell populations. FHIT has been widely investigated in cancer cells, but to our knowledge this is the first direct evidence of spontaneous and recurrent occurrence of genomic instability at this gene in human subjects, at the same region involved in cancer rearrangements. Our results suggest that common fragile site activity is not restricted to in vitro cell culture and that genomic instability may pre-exist in normal cells in the absence of exogenous replication stress.

Replication dynamics at common fragile site FRA6E.

The replication dynamics at common fragile site FRA6E has been evaluated by molecular combing and interphase fluorescent in situ hybridisation (FISH) in primary human lymphocytes cultured under normal or aphidicolin-induced stress conditions. FRA6E is one of the most frequently expressed common fragile sites of the human genome. It harbours several genes, PARK2 being regarded as the most relevant one. According to the results obtained from interphase FISH analysis, FRA6E can be considered a mid-late-replicating sequence characterised by heterogeneous replication timing. Molecular combing did not reveal specific replication parameters at the fragile site: fork rates were highly comparable to those detected at an early replicating locus (LMNB2) used as control and in very good agreement with the whole-genome data obtained in parallel. The same indication applied to the density of initiation zones, the inter-origin distances from adjacent ongoing forks, the frequencies of unidirectional forks, fork arrest events and asynchronous forks. Interestingly, PARK2 appeared embedded in an early/late replication transition zone, corresponding to intron 8 (162 kb) and to the fragility core of FRA6E. In cells exposed to aphidicolin, few forks progressing at a rather slow rate were observed, the majority of them being unidirectional, but again a specific response of the fragile site was not observed. In summary, at FRA6E the replication process is not impaired per se, but chromosome breakages occur preferentially at an early/late replication transition zone. Aphidicolin might increase the occurrence of breakage events at FRA6E by prolonging the time interval separating the replication of early and late replication domains. These results may be of general significance to address the problem of fragile site instability.