Precision analysis of mutant U2AF1 activity reveals deployment of stress granules in myeloid malignancies.

Splicing factor mutations are common among cancers, recently emerging as drivers of myeloid malignancies. U2AF1 carries hotspot mutations in its RNA-binding motifs; however, how they affect splicing and promote cancer remain unclear. The U2AF1/U2AF2 heterodimer is critical for 3' splice site (3'SS) definition. To specifically unmask changes in U2AF1 function in vivo, we developed a crosslinking and immunoprecipitation procedure that detects contacts between U2AF1 and the 3'SS AG at single-nucleotide resolution. Our data reveal that the U2AF1 S34F and Q157R mutants establish new 3'SS contacts at -3 and +1 nucleotides, respectively. These effects compromise U2AF2-RNA interactions, resulting predominantly in intron retention and exon exclusion. Integrating RNA binding, splicing, and turnover data, we predicted that U2AF1 mutations directly affect stress granule components, which was corroborated by single-cell RNA-seq. Remarkably, U2AF1-mutant cell lines and patient-derived MDS/AML blasts displayed a heightened stress granule response, pointing to a novel role for biomolecular condensates in adaptive oncogenic strategies.

SR proteins are NXF1 adaptors that link alternative RNA processing to mRNA export.

Nuclear export factor 1 (NXF1) exports mRNA to the cytoplasm after recruitment to mRNA by specific adaptor proteins. How and why cells use numerous different export adaptors is poorly understood. Here we critically evaluate members of the SR protein family (SRSF1-7) for their potential to act as NXF1 adaptors that couple pre-mRNA processing to mRNA export. Consistent with this proposal, >1000 endogenous mRNAs required individual SR proteins for nuclear export in vivo. To address the mechanism, transcriptome-wide RNA-binding profiles of NXF1 and SRSF1-7 were determined in parallel by individual-nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP). Quantitative comparisons of RNA-binding sites showed that NXF1 and SR proteins bind mRNA targets at adjacent sites, indicative of cobinding. SRSF3 emerged as the most potent NXF1 adaptor, conferring sequence specificity to RNA binding by NXF1 in last exons. Interestingly, SRSF3 and SRSF7 were shown to bind different sites in last exons and regulate 3' untranslated region length in an opposing manner. Both SRSF3 and SRSF7 promoted NXF1 recruitment to mRNA. Thus, SRSF3 and SRSF7 couple alternative splicing and polyadenylation to NXF1-mediated mRNA export, thereby controlling the cytoplasmic abundance of transcripts with alternative 3' ends.

Interaction of miR-155 with Human Serum Albumin: An Atomic Force Spectroscopy, Fluorescence, FRET, and Computational Modelling Evidence.

This study investigated the interaction between Human Serum Albumin (HSA) and microRNA 155 (miR-155) through spectroscopic, nanoscopic and computational methods. Atomic force spectroscopy together with static and time-resolved fluorescence demonstrated the formation of an HSA/miR-155 complex characterized by a moderate affinity constant (KA in the order of 104 M-1). Förster Resonance Energy Transfer (FRET) experiments allowed us to measure a distance of (3.9 ± 0.2) nm between the lone HSA Trp214 and an acceptor dye bound to miR-155 within such a complex. This structural parameter, combined with computational docking and binding free energy calculations, led us to identify two possible models for the structure of the complex, both characterized by a topography in which miR-155 is located within two positively charged pockets of HSA. These results align with the interaction found for HSA and miR-4749, reinforcing the thesis that native HSA is a suitable miRNA carrier under physiological conditions for delivering to appropriate targets.

Interaction between miR4749 and Human Serum Albumin as Revealed by Fluorescence, FRET, Atomic Force Spectroscopy and Computational Modelling.

The interaction of Human Serum Albumin (HSA) with the microRNA, miR4749, was investigated by Atomic Force Spectrscopy (AFS), static and time-resolved fluorescence spectroscopy and by computational methods. The formation of a HSA/miR4749 complex with an affinity of about 104 M-1 has been assessed through a Stern-Volmer analysis of steady-state fluorescence quenching of the lone Trp residue (Trp214) emission of HSA. Förster Resonance Energy Transfer (FRET) measurements of fluorescence lifetime of the HSA/miR4749 complex were carried out in the absence and in the presence of an acceptor chromophore linked to miR4749. This allowed us to determine a distance of 4.3 ± 0.5 nm between the lone Trp of HSA and the dye bound to miR4749 5p-end. Such a distance was exploited for a screening of the possible binding sites between HSA and miR4749, as predicted by computational docking. Such an approach, further refined by binding free energy calculations, led us to the identification of a consistent model for the structure of the HSA/miR4749 complex in which a positively charged HSA pocket accommodates the negatively charged miRNA molecule. These results designate native HSA as a suitable miRNA carrier under physiological conditions for delivering to appropriate targets.

Amphiphilicity-Controlled Localization of Red Emitting Bicationic Fluorophores in Tumor Cells Acting as Bio-Probes and Anticancer Drugs.

Small organic molecules arouse lively interest for their plethora of possible biological applications, such as anticancer therapy, for their ability to interact with nucleic acids, or bioimaging, thanks to their fluorescence emission. Here, a panchromatic series of styryl-azinium bicationic dyes, which have already proved to exhibit high water-solubility and significant red fluorescence in water, were investigated through spectrofluorimetric titrations to assess the extent of their association constants with DNA and RNA. Femtosecond-resolved transient absorption spectroscopy was also employed to characterize the changes in the photophysical properties of these fluorophores upon interaction with their biological targets. Finally, in vitro experiments conducted on tumor cell lines revealed that some of the bicationic fluorophores had a peculiar localization within cell nuclei exerting important antiproliferative effects, others were instead found to localize in the cytoplasm without leading to cell death, being useful to mark specific organelles in light of live cell bioimaging. Interestingly, this molecule-dependent behavior matched the different amphiphilicity featured by these bioactive compounds, which are thus expected to be caught in a tug-of-war between lipophilicity, ensured by the presence of aromatic rings and needed to pass cell membranes, and hydrophilicity, granted by charged groups and necessary for stability in aqueous media.

Quaternized styryl-azinium fluorophores as cellular RNA-binders.

The capability of three quaternized styryl-azinium iodides to bind cellular RNA has been tested by means of Fluorescence Confocal Microscopy imaging of stained MCF-7 cells treated with RNase. Their association constants have been estimated through spectrophotometric and fluorimetric titrations with tRNA and compared to their affinity toward DNA. Transient absorption spectroscopy with femtosecond resolution confirmed the binding of the investigated compounds with tRNA and shed new light on the excited state dynamics of their complexes, by revealing a significant lengthening of the lifetime of S1 upon complexation, which parallels the fluorescence quantum yield enhancement.

Fine structural tuning of styryl-based dyes for fluorescence and CD-based sensing of various ds-DNA/RNA sequences.

A set of styryl- and bis-styryl dyes, varying in length, aromatic surface, net positive charge and steric positioning or bulkiness of substituents, was tested for interactions with various ds-DNA or ds-RNA. Most of the compounds showed strong affinity toward ds-DNA/RNA, directly correlated to the synergistic contribution of the aromatic-conjugated surface and net positive charge. The volume or positioning of terminal aromatic substituents directly controlled the binding mode of the core structure, shifting between DNA/RNA groove binding or DNA/RNA intercalation. Consequently, upon binding to DNA/RNA the fluorimetric and induced CD (ICD) response varied for different compounds, for instance one derivative showed specific fluorescence increase with AT-DNA, while another derivative showed specific ICD response with AU-RNA. Preliminary screening on human tumour cell lines revealed an efficient cellular uptake for all dyes. Only mono-styryl-quinoline derivatives showed a strong antiproliferative activity combined with efficient fluorescent localisation, thus showing promising theragnostic potential, while other compounds were negligibly cytotoxic but still efficient fluorescent markers of cytoplasmic organelles.

Fractionation iCLIP detects persistent SR protein binding to conserved, retained introns in chromatin, nucleoplasm and cytoplasm.

RNA binding proteins (RBPs) regulate the lives of all RNAs from transcription, processing, and function to decay. How RNA-protein interactions change over time and space to support these roles is poorly understood. Towards this end, we sought to determine how two SR proteins-SRSF3 and SRSF7, regulators of pre-mRNA splicing, nuclear export and translation-interact with RNA in different cellular compartments. To do so, we developed Fractionation iCLIP (Fr-iCLIP), in which chromatin, nucleoplasmic and cytoplasmic fractions are prepared from UV-crosslinked cells and then subjected to iCLIP. As expected, SRSF3 and SRSF7 targets were detected in all fractions, with intron, snoRNA and lncRNA interactions enriched in the nucleus. Cytoplasmically-bound mRNAs reflected distinct functional groupings, suggesting coordinated translation regulation. Surprisingly, hundreds of cytoplasmic intron targets were detected. These cytoplasmic introns were found to be highly conserved and introduced premature termination codons into coding regions. However, many intron-retained mRNAs were not substrates for nonsense-mediated decay (NMD), even though they were detected in polysomes. These findings suggest that intron-retained mRNAs in the cytoplasm have previously uncharacterized functions and/or escape surveillance. Hence, Fr-iCLIP detects the cellular location of RNA-protein interactions and provides insight into co-transcriptional, post-transcriptional and cytoplasmic RBP functions for coding and non-coding RNAs.

Detection of miR-155 Using Peptide Nucleic Acid at Physiological-like Conditions by Surface Plasmon Resonance and Bio-Field Effect Transistor.

MicroRNAs are small ribonucleotides that act as key gene regulators. Their altered expression is often associated with the onset and progression of several human diseases, including cancer. Given their potential use as biomarkers, there is a need to find detection methods for microRNAs suitable for use in clinical setting. Field-effect-transistor-based biosensors (bioFETs) appear to be valid tools to detect microRNAs, since they may reliably quantitate the specific binding between the immobilized probe and free target in solution through an easily detectable electrical signal. We have investigated the detection of human microRNA 155 (miR-155) using an innovative capturing probe constituted by a synthetic peptide nucleic acid (PNA), which has the advantage to form a duplex even at ionic strengths approaching the physiological conditions. With the aim to develop an optimized BioFET setup, the interaction kinetics between miR-155 and the chosen PNA was preliminarily investigated by using surface plasmon resonance (SPR). By exploiting both these results and our custom-made bioFET system, we were able to attain a low-cost, real-time, label-free and highly specific detection of miR-155 in the nano-molar range.

Nanoscale dynamical investigation of the hemoglobin complex with the bacterial protein IsdB: is their interaction stabilized by catch bonds?

Iron-regulated surface determinant B (IsdB) is a surface protein of Staphylococcus aureus that plays essential roles in host cell invasion by mediating both bacterial adhesion and hemic iron acquisition. Single-molecule experiments have recently revealed that the binding of IsdB to vitronectin and integrins is dramatically strengthened under mechanical stress conditions, promoting staphylococcal adhesion. Here we conducted atomic force spectroscopy (AFS) measurements of the interaction between IsdB and hemoglobin (Hb), in both its oxidized (metHb) and reduced forms (HbCO). While the former represents the natural substrate for IsdB, the latter is resistant to heme extraction. For the unbinding between IsdB and HbCO, we obtained a linear trend in the Bell-Evans plot, indicative of a weakening of the interaction upon mechanical stress. For the unbinding between IsdB and metHb, we found similar behavior at low loading rates. Remarkably, a non-linear trend of the complex interaction force was detected at higher force-pulling rates. Such behavior may provide some cues to the ability of IsdB to form stress-dependent bonds also with Hb, possibly enabling a more efficient heme transfer through stabilization of the transient (in vivo) IsdB-Hb complex.

Hybridization Kinetics of miR-155 on Gold Surfaces as Investigated by Surface Plasmon Resonance and Atomic Force Spectroscopy.

miRNAs are short noncoding RNA single strands, with a crucial role in several biological processes. miRNAs are dysregulated in several human diseases, and their detection is an important goal for diagnosis and screening. Innovative biosensors for miRNAs are commonly based on the hybridization process between a miRNA and its corresponding complementary strand (or suitable aptamers) immobilized onto an electrode surface forming a duplex. A detailed description of the hybridization kinetics in working conditions deserves a great deal of interest for the optimization of the biosensing process. Surface plasmon resonance (SPR) and atomic force spectroscopy (AFS) were applied to investigate the hybridization process between miR-155, a multifunctional miRNA that constitutes an important marker overexpressed in several diseases, and its complementary strand (antimiR-155), immobilized on the gold-coated surface of a commercial electrode. Under well-adjusted pH, ionic strength, surface coverage, and concentration, we found that miR-155 has a high affinity for antimiR-155 with kinetics well described by the 1:1 Langmuir model. Both techniques provided an association rate of about 104 M-1 s-1, while a dissociation rate of 10-5 and 10-4 s-1 was assessed by SPR and AFS, respectively. These results allowed us to establish optimized measurement running times for applications in biosensing. An analysis of AFS data also led us to evaluate the binding free energy for the duplex, which was found to be close to that of free molecules in solution. These results could guide in the implementation of fine-tuned working conditions of a biosensor for detecting miRNAs based on correspondent complementary strands.

Integrative genome-wide analysis reveals EIF3A as a key downstream regulator of translational repressor protein Musashi 2 (MSI2).

Musashi 2 (MSI2) is an RNA binding protein (RBP) that regulates asymmetric cell division and cell fate decisions in normal and cancer stem cells. MSI2 appears to repress translation by binding to 3' untranslated regions (3'UTRs) of mRNA, but the identity of functional targets remains unknown. Here, we used individual nucleotide resolution cross-linking and immunoprecipitation (iCLIP) to identify direct RNA binding partners of MSI2 and integrated these data with polysome profiling to obtain insights into MSI2 function. iCLIP revealed specific MSI2 binding to thousands of mRNAs largely in 3'UTRs, but translational differences were restricted to a small fraction of these transcripts, indicating that MSI2 regulation is not triggered by simple binding. Instead, the functional targets identified here were bound at higher density and contain more 'UAG' motifs compared to targets bound nonproductively. To further distinguish direct and indirect targets, MSI2 was acutely depleted. Surprisingly, only 50 transcripts were found to undergo translational induction on acute loss. Using complementary approaches, we determined eukaryotic translation initiation factor 3A (EIF3A) to be an immediate, direct target. We propose that MSI2 downregulation of EIF3A amplifies these effects on translation. Our results also underscore the challenges in defining functional targets of RBPs since mere binding does not imply a discernible functional interaction.

Cellular differentiation state modulates the mRNA export activity of SR proteins.

SR proteins function in nuclear pre-mRNA processing, mRNA export, and translation. To investigate their cellular dynamics, we developed a quantitative assay, which detects differences in nucleocytoplasmic shuttling among seven canonical SR protein family members. As expected, SRSF2 and SRSF5 shuttle poorly in HeLa cells but surprisingly display considerable shuttling in pluripotent murine P19 cells. Combining individual-resolution cross-linking and immunoprecipitation (iCLIP) and mass spectrometry, we show that elevated arginine methylation of SRSF5 and lower phosphorylation levels of cobound SRSF2 enhance shuttling of SRSF5 in P19 cells by modulating protein-protein and protein-RNA interactions. Moreover, SRSF5 is bound to pluripotency-specific transcripts such as Lin28a and Pou5f1/Oct4 in the cytoplasm. SRSF5 depletion reduces and overexpression increases their cytoplasmic mRNA levels, suggesting that enhanced mRNA export by SRSF5 is required for the expression of pluripotency factors. Remarkably, neural differentiation of P19 cells leads to dramatically reduced SRSF5 shuttling. Our findings indicate that posttranslational modification of SR proteins underlies the regulation of their mRNA export activities and distinguishes pluripotent from differentiated cells.

The hepatitis C virus E1 glycoprotein undergoes productive folding but accelerated degradation when expressed as an individual subunit in CHO cells.

Hepatitis C Virus E1E2 heterodimers are components of the viral spike. Although there is a general agreement on the necessity of the co-expression of both E1 and E2 on a single coding unit for their productive folding and assembly, in a previous study using an in vitro system we obtained strong indications that E1 can achieve folding in absence of E2. Here, we have studied the folding pathway of unescorted E1 from stably expressing CHO cells, compared to the folding observed in presence of the E2 protein. A DTT-resistant conformation is achieved by E1 in both situations, consistent with the presence of an E2-independent oxidative pathway. However, while the E1E2 heterodimer is stable inside cells, E1 expressed alone is degraded within a few hours. On the other hand, the oxidation and stability of individually expressed E2 subunits is dependent on E1 co-expression. These data are consistent with E1 and E2 assisting each other for correct folding via different mechanisms: E2 assists E1 by stabilizing a semi-native conformation meanwhile E1 drives E2 towards a productive folding pathway.