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Medium-sized ncRNAs in diseases

Massive parallel sequencing technologies revealed that large fractions of eukaryotic genomes are transcribed. Interestingly, just a portion of the transcriptome seems to be protein-coding. Many transcripts are considered to encode no protein and are thus referred to as non-coding RNA (ncRNA). Among those ncRNAs exist RNA species with a size of 50 to 300 nts in length. These are referred to as medium-sized ncRNAs (msRNAs) in contrast to long ncRNAs (lncRNAs; >200 nts) or small ncRNAs (e.g. miRNAs/piRNAs; <50 nts). Although msRNAs are quite heterogeneous in respect to their biogenesis, cellular localization and functions it is generally accepted that many of these RNAs serve essential cellular roles like in translation, transcription or splicing. Due to the variety of processes controlled by msRNAs it is important to characterize the expression patterns of msRNAs and how they contribute to cell functions in health and disease.

Our lab focuses on the identification and characterization of msRNAs. Therefore we use a combination of msRNA-purification protocols combined with RNA sequencing to determine msRNA expression in a given cell type or tissue (msRNAseq). Relevant msRNAs are then characterized by a number of methods including Northern Blotting, fluorescence in situ hybridization (FISH) or glycerol gradient analyses. The lab was established as a collaborative junior group of the institutes of molecular medicine (IMM) and physiology (JBI). With utilizing the resources provided by both institutes we address the role of msRNAs in neoplastic (IMM) and cardiovascular (JBI) diseases. For questions and suggestions please contact our staff or visit the website of the msRNAdb found here. 

 

The role of APOBEC proteins in renal cell carcinoma

 

Kidney cancer is one of the most common cancer types affecting nearly 10.000 people in Germany every year. The most common type of kidney cancer is the clear cell renal cell carcinoma (ccRCC). While prognosis for low stage ccRCCs is quite favorable (60-80% 5-year-survival rate), metastatic ccRCC is characterized by very low survival rates. Since most RCCs are resistant to classical chemo- and radiotherapies, novel efficient therapies especially targeting metastasizing RCCs are needed.

 

 

We analyzed the molecular signature of RCC subtypes in TCGA data sets. Surprisingly, we found members of the APOBEC3 family of RNA-binding proteins (RBPs) to be significantly upregulated in ccRCCs. APOBEC3 proteins belong to a family of Zn-dependent cytidine deaminases. Primate APOBEC3 genes underwent gene duplication events that led to the diversification of this gene family with seven members in humans. We focused our research so far on the two family members APOBEC3C (A3C) and APOBEC3G (A3G). While their role as antiviral RBPs and editing enzymes has been well investigated in hematopoietic cells, there role in other cell types is poorly understood. APOBEC3 proteins were shown to associate with a variety of medium-sized ncRNAs (msRNAs) although the functional role of this has not been determined. Currently we are establishing RCC-derived cell lines either overexpressing A3C/G or that exhibit CRISPR/Cas9 mediated downregulation of both proteins. We expect that the modulation of APOBEC3 proteins significantly alters tumor cell properties in RCC-derived cell lines. Since the interferon pathway plays an important role in RCC progression and A3C/G are interferon responsive genes we aim at the further characterization of the role A3C/G in the modulation of this pathway. Since interferon alpha therapy is conducted for treatment of metastatic RCCs we also expect that our research will shed light on the role of this pathway on RCC progression and therapy.

Rbfox1 – a hub controlling splicing and ncRNA fate in cardiovascular disease

The RBFOX-family of RNA-binding proteins (RBPs) constitutes a group of conserved regulators of alternative splicing (AS). RBFOX1 is mainly expressed in neurons and muscle tissue. This expression pattern is achieved by the use of alternative promoters and AS resulting in neuronal and muscular isoforms. Animal models confirmed the essential role of RBFOX1 in the regulation of alternative splicing. Hence, it was described that RBFOX1 is strongly decreased following transverse aortic constriction (TAC) in mice. In this model of cardiac hypertrophy several splicing changes have been observed, which could be partially explained by the loss of RBFOX1 expression. These studies suggested that a decrease in RBFOX1 expression correlates with pathologies that involve a progressive muscle de-differentiation (e.g. cardiac hypertrophy or muscle dystrophy). In addition to AS, RBFOX-variants had been implicated in the regulation of miRNA-biogenesis as well as influencing the processing of snoRNAs due to the association with a conserved GCAUG-motif in several precursor ncRNAs. So far, there is just limited information about the mechanisms underlying the de-regulation of RBFOX1-expression. 

 

 

We focused on the characterization of RBFOX1 in the heart and used a knockout mouse model with genetically induced severe heart hypertrophy without heart failure established in the JBI (EGFRΔ/ΔVSMC&CM, Schreier et al., 2013). We confirmed that in addition to the previously mentioned TAC-model, RBFOX1 expression is also strongly reduced in hypertrophic hearts of these animals. We currently analyze the muscle specific target spectrum of RBFOX1 to identify phenotypes that are caused by RBFOX1 depletion in the failing heart. We consider RBFOX1 as a hub controlling proteins that regulate cell morphology, actin dynamics and contractility especially in the context of cardiovascular diseases. A decrease in RBFOX1 expression therefore leads to splicing changes, which promote cardiovascular pathologies as seen in models of cardiac hypertrophy.