Ionut Atanasoai

Objective To better comprehend transcriptional phenotypes of cancer cells, we globally characterised RNA-binding proteins (RBPs) to identify altered RNAs, including long non-coding RNAs (lncRNAs). Design To unravel RBP-lncRNA interactions in cancer, we curated a list of ~2300 highly expressed RBPs in human cells, tested effects of RBPs and lncRNAs on patient survival in multiple cohorts, altered expression levels, integrated various sequencing, molecular and cell-based data. Results High expression of RBPs negatively affected patient survival in 21 cancer types, especially hepatocellular carcinoma (HCC). After knockdown of the top 10 upregulated RBPs and subsequent transcriptome analysis, we identified 88 differentially expressed lncRNAs, including 34 novel transcripts. CRISPRa-mediated overexpression of four lncRNAs had major effects on the HCC cell phenotype and transcriptome. Further investigation of four RBP-lncRNA pairs revealed involvement in distinct regulatory processes. The most noticeable RBP-lncRNA connection affected lipid metabolism, whereby the non-canonical RBP CCT3 regulated LINC00326 in a chaperonin-independent manner. Perturbation of the CCT3- LINC00326 regulatory network led to decreased lipid accumulation and increased lipid degradation in cellulo as well as diminished tumour growth in vivo . Conclusions We revealed that RBP gene expression is perturbed in HCC and identified that RBPs exerted additional functions beyond their tasks under normal physiological conditions, which can be stimulated or intensified via lncRNAs and affected tumour growth.

With the rise of new powerful genome engineering technologies, such as CRISPR/Cas9, cell models can be engineered effectively to accelerate basic and disease research. The most critical step in this procedure is the efficient delivery of foreign nucleic acids into cells by cellular transfection. Since the vectors encoding the components necessary for CRISPR/Cas genome engineering are always large (9-19 kb), they result in low transfection efficiency and cell viability, and thus subsequent selection or purification of positive cells is required. To overcome those obstacles, we here show a non-toxic and non-viral delivery method that increases transfection efficiency (up to 40-fold) and cell viability (up to 6-fold) in a number of hard-to-transfect human cancer cell lines and primary blood cells. At its core, the technique is based on adding exogenous small plasmids of a defined size to the transfection mixture.

SUMMARY Over the past decade, thousands of putative human RNA binding proteins (RBPs) have been identified and increased the demand for specifying RNA binding capacities. Here, we developed RNA affinity purification followed by sequencing (RAPseq) that enables in vitro large-scale profiling of RBP binding to native RNAs. First, by employing RAPseq, we found that vertebrate HURs recognize a conserved RNA binding motif and bind predominantly to introns in zebrafish compared to 3’UTRs in human RNAs. Second, our dual RBP assays (co-RAPseq) uncovered cooperative RNA binding of HUR and PTBP1 within an optimal distance of 27 nucleotides. Third, we developed T7-RAPseq to discern m 6 A-dependent and - independent RNA binding sites of YTHDF1. Fourth, RAPseq of 26 novel non-canonical RBPs revealed specialized moonlighting interactions. Last, five pathological IGF2BP family variants exhibited different RNA binding patterns. Overall, our simple, scalable and versatile method enables to fast-forward RBP-related questions. Graphical Abstract HIGHLIGHTS RAPseq reveals in vitro -derived RBP-RNA interactomes the vertebrate-conserved HUR binding motif adapted to species-unique RNA features co-RAPseq and T7-RAPseq uncover binding cooperativity and modification dependencies non-canonical RBPs have specialized RNA interactomes

Abstract The pluripotent state is not solely governed by the action of the core transcription factors Oct4, Sox2, and Nanog, but also by a series of co-transcriptional and post-transcriptional events, including alternative splicing (AS) and the interaction of RNA-binding proteins (RBPs) with defined subpopulations of RNAs. Zinc Finger Protein 207 (ZFP207) is an essential transcription factor for mammalian embryonic development. Here, we employ multiple functional analyses to characterize the role of ZFP207 in mouse embryonic stem cells (ESCs). We find that ZFP207 plays a pivotal role in ESC maintenance, and silencing of Zfp207 leads to severe neuroectodermal differentiation defects. In striking contrast to human ESCs, ZFP207 does not transcriptionally regulate stem cell and neuronal-related genes but exerts its effects by control AS networks and acting as an RBP. Our study expands the role of ZFP207 to maintain ESC identity, and underscores ZFP207 functional versatility with key roles in neural fate commitment.

Background: ) copy number, resulting in elevated serum tryptase levels. Although often asymptomatic, HαT is associated with anaphylaxis, flushing, and connective tissue abnormalities. Although mast cells are primarily implicated, basophil involvement in HαT remains poorly defined. Objective: Our aim was to compare basophil proportions, MRGPRX2 expression, and responsiveness to IgE-dependent and IgE-independent activation in individuals with HαT, individuals with indolent systemic mastocytosis (ISM), and healthy controls (HCs). Methods: -formylmethionyl-leucyl-phenylalanine, anti-FcεRI antibody, mastoparan, and compound 48/80. Results: Relative basophil proportions were higher in subjects with HαT than in subjects with ISM. FcεRI surface expression was preserved in those with HαT but reduced in those with ISM, whereas MRGPRX2 expression was not detected at functionally relevant levels. Basophils from individuals with HαT displayed nonresponsiveness to anti-FcεRI more frequently. In contrast, response to formylmethionyl-leucyl-phenylalanine was higher in subjects with ISM than in subjects with HαT and showed a trend of being higher than in HCs. Mastoparan- and compound 48/80-induced activation was undetectable across groups. Conclusion: HαT features enriched basophil frequency but lacks functional hyperreactivity. An increased rate of nonresponse to FcεRI cross-linking distinguishes HαT from ISM, indicating condition-specific FcεRI signaling dysregulation rather than uniform basophil dysfunction in mast cell-associated disorders.