Dual role of Lin28a in the regulation of miRNA biogenesis during neuronal differentiation

Many cellular functions depend on the tightly regulated expression of various proteins. Canonical control of the protein expression is associated with transcriptional regulation. However, the small non-coding RNAs called microRNAs (miRNAs) were identified as post-transcriptional regulators of gene expression. In a typical manner, miRNAs originate similarly to the coding RNAs and are processed in a multi-step maturation process. It has been shown that miRNAs are very important for the proper functioning of tissues. Interestingly, the human nervous system contains over 70% of all miRNAs; thus, the maturation process has to be tightly regulated. However, despite the important role of miRNAs, little is known about the mechanisms regulating their biogenesis. In my PhD project, I showed that during early stages of neuronal differentiation, Lin28a controls levels of neuro-specific miRNA-9. I demonstrated that Lin28a binds to the conserved terminal loop (CTL) of pre-miRNA-9 and decreases the cellular levels of miRNA-9 during retinoic acid-mediated neuronal differentiation of mouse teratocarcinoma P19 cells. I revealed that the Lin28a-mediated inhibition of miRNA-9 production was uridylation-independent. Furthermore, constitutive expression of GFP-tagged Lin28a reduced the levels of let-7a but not miRNA-9, whereas untagged Lin28a inhibited both miR-9 and let-7a during the course of neuronal differentiation. Using small RNAseq analysis of P19 cells with constitutive expression of Lin28a I showed that it controls many more miRNAs than previously recognised. Intriguingly, many miRNAs were upregulated by Lin28a overexpression. I demonstrated with high-throughput, the limited function of GFP-tagged Lin28a results, and I also showed that untagged Lin28a inhibits the production of a number of brain-specific miRNAs including miRNA-9. Finally, I revealed that 3’-5’exoribonuclease Dis3l2 was responsible for uridylation-independent degradation of pre-miRNA-9. Altogether, my results provided evidence that Lin28a has both positive and negative roles in the regulation of miRNA production and has a dual role in triggering pre-miRNA degradation

Lin28a uses distinct mechanisms of binding to RNA and affects positively and negatively miRNA levels

Lin28a inhibits the biogenesis of let-7 miRNAs by triggering the polyuridylation and degradation of their precursors by terminal uridylyltransferases TUT4/7 and 3′-5′ exoribonuclease Dis3l2, respectively. Previously, we showed that Lin28a also controls the production of neuro-specific miRNA-9 via a polyuridylation-independent mechanism. Here we reveal that the sequences and structural characteristics of pre-let-7 and pre-miRNA-9 are eliciting two distinct modes of binding to Lin28a. We present evidence that Dis3l2 controls miRNA-9 production. Finally, we show that the constitutive expression of untagged Lin28a during neuronal differentiation in vitro positively and negatively affects numerous other miRNAs. Our findings shed light on the role of Lin28a in differentiating cells and on the ways in which one RNA-binding protein can perform multiple roles in the regulation of RNA processing

Grazing angle X-ray fluorescence from periodic structures on silicon and silica surfaces

Various 3-dimensional nano-scaled periodic structures with different configurations and periods deposited on the surface of silicon and silica substrates were investigated by means of the grazing incidence and grazing emission X-ray fluorescence techniques. Apart from the characteristics which are typical for particle- and layer-like samples, the measured angular intensity profiles show additional periodicity-related features. The latter could be explained by a novel theoretical approach based on simple geometrical optics (GO) considerations. The new GO-based calculations were found to yield results in good agreement with experiment, also in cases where other theoretical approaches are not valid, e.g., periodic particle distributions with an increased surface coverage

RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain and is required for ubiquitination

Background TRIM25 is a novel RNA-binding protein and a member of the Tripartite Motif (TRIM) family of E3 ubiquitin ligases, which plays a pivotal role in the innate immune response. However, there is scarce knowledge about its RNA-related roles in cell biology. Furthermore, its RNA-binding domain has not been characterized. Results Here, we reveal that the RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain, which we postulate to be a novel RNA-binding domain. Using CLIP-seq and SILAC-based co-immunoprecipitation assays, we uncover TRIM25’s endogenous RNA targets and protein binding partners. We demonstrate that TRIM25 controls the levels of Zinc Finger Antiviral Protein (ZAP). Finally, we show that the RNA-binding activity of TRIM25 is important for its ubiquitin ligase activity towards itself (autoubiquitination) and its physiologically relevant target ZAP. Conclusions Our results suggest that many other proteins with the PRY/SPRY domain could have yet uncharacterized RNA-binding potential. Together, our data reveal new insights into the molecular roles and characteristics of RNA-binding E3 ubiquitin ligases and demonstrate that RNA could be an essential factor in their enzymatic activity

Interleukin-6 as a Predictive Factor of Pathological Response to FLOT Regimen Systemic Treatment in Locally Advanced Gastroesophageal Junction or Gastric Cancer Patients

Background: Perioperative treatment is a gold standard in locally advanced gastric cancer or GEJ cancer in the Western population. Unfortunately, the response rate after neoadjuvant chemotherapy (NAC) remains limited. Moreover, there are currently no biomarkers enabling an individual prediction of therapeutic efficacy. The aim of this study was the identification of serum biomarkers of early response to NAC. Methods: We conducted this prospective study in the MSCNRIO in Warsaw, Poland. A total of 71 patients and 15 healthy volunteers gave informed consent. Complete blood count, carcinoembryonic antigen (CEA), carcinoma antigen 125 (CA125), carcinoma antigen 19.9 (CA19.9), and fibrinogen (F) were measured at baseline and before every cycle. Circulating tumour cells (CTCs) and interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin-10 (IL-10) were measured in a pilot group of 40 patients at baseline and before cycle two (C2) and cycle three (C3). Results: Of all the measured parameters, only the IL-6 serum level was statistically significant. The IL-6 level before C2 of chemotherapy was significantly decreased in the complete pathological response (pCR) vs. the non-pCR group (3.71 pg/mL vs. 7.63 pg/mL, p = 0.004). In all patients with an IL-6 level below 5.0 pg/mL in C2, tumour regression TRG1a/1b according to the Becker classification and ypN0 were detected in postoperative histopathological specimens. The IL-6 level before C1 of chemotherapy was significantly elevated in ypN+ vs. ypN0 (7.69 pg/mL vs. 2.89 pg/mL, p = 0.022). Conclusions: The trial showed that an elevated level of IL-6 prior to treatment and C2 might be a predictor of pathological response to NAC