Small but Powerful: The Human Vault RNAs as Multifaceted Modulators of Pro-Survival Characteristics and Tumorigenesis.

The importance of non-coding RNAs for regulating gene expression has been uncovered in model systems spanning all three domains of life. More recently, their involvement in modulating signal transduction, cell proliferation, tumorigenesis and cancer progression has also made them promising tools and targets for oncotherapy. Recent studies revealed a class of highly conserved small ncRNAs, namely vault RNAs, as regulators of several cellular homeostasis mechanisms. The human genome encodes four vault RNA paralogs that share significant sequence and structural similarities, yet they seem to possess distinct roles in mammalian cells. The alteration of vault RNA expression levels has frequently been observed in cancer tissues, thus hinting at a putative role in orchestrating pro-survival characteristics. Over the last decade, significant advances have been achieved in clarifying the relationship between vault RNA and cellular mechanisms involved in cancer development. It became increasingly clear that vault RNAs are involved in controlling apoptosis, lysosome biogenesis and function, as well as autophagy in several malignant cell lines, most likely by modulating signaling pathways (e.g., the pro-survival MAPK cascade). In this review, we discuss the identified and known functions of the human vault RNAs in the context of cell proliferation, tumorigenesis and chemotherapy resistance.

The human vault RNA enhances tumorigenesis and chemoresistance through the lysosome in hepatocellular carcinoma.

The small non-coding VTRNA1-1 (vault RNA 1-1) is known to confer resistance to apoptosis in several malignant cell lines and to also modulate the macroautophagic/autophagic flux in hepatocytes, thus highlighting its pro-survival role. Here we describe a new function of VTRNA1-1 in regulating in vitro and in vivo tumor cell proliferation, tumorigenesis and chemoresistance. Knockout (KO) of VTRNA1-1 in human hepatocellular carcinoma cells reduced nuclear localization of TFEB (transcription factor EB), leading to a downregulation of the coordinated lysosomal expression and regulation (CLEAR) network genes and lysosomal compartment dysfunction. We demonstrate further that impaired lysosome function due to loss of VTRNA1-1 potentiates the anticancer effect of conventional chemotherapeutic drugs. Finally, loss of VTRNA1-1 reduced drug lysosomotropism allowing higher intracellular compound availability and thereby significantly reducing tumor cell proliferation in vitro and in vivo. These findings reveal a so far unknown role of VTRNA1-1 in the intracellular catabolic compartment and describe its contribution to lysosome-mediated chemotherapy resistance.Abbreviations: ATP6V0D2: ATPase H+ transporting V0 subunit d2; BafA: bafilomycin A1; CLEAR: coordinated lysosomal expression and regulation; CQ: chloroquine; DMSO: dimethyl sulfoxide; GST-BHMT: glutathionine S-transferase N-terminal to betaine-homocysteine S-methyltransferase; HCC: hepatocellular carcinoma; LAMP1: lysosomal associated membrane protein 1; LLOMe: L-leucyl-L-leucine methyl ester; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MAPK: mitogen-activated protein kinase; MITF: melanocyte inducing transcription factor; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ncRNA: non-coding RNA; RNP: ribonucleoprotein; SF: sorafenib; SQSTM1/p62: sequestosome 1; STS: staurosporine; tdRs: tRNA-derived RNAs; TFE3: transcription factor binding to IGHM enhancer 3; TFEB: transcription factor EB; vtRNA: vault RNA transcript.

Human vtRNA1-1 Levels Modulate Signaling Pathways and Regulate Apoptosis in Human Cancer Cells.

Regulatory non-protein coding RNAs perform a remarkable variety of complex biological functions. Previously, we demonstrated a role of the human non-coding vault RNA1-1 (vtRNA1-1) in inhibiting intrinsic and extrinsic apoptosis in several cancer cell lines. Yet on the molecular level, the function of the vtRNA1-1 is still not fully clear. Here, we created HeLa knock-out cell lines revealing that prolonged starvation triggers elevated levels of apoptosis in the absence of vtRNA1-1 but not in vtRNA1-3 knock-out cells. Next-generation deep sequencing of the mRNome identified the PI3K/Akt pathway and the ERK1/2 MAPK cascade, two prominent signaling axes, to be misregulated in the absence of vtRNA1-1 during starvation-mediated cell death conditions. Expression of vtRNA1-1 mutants identified a short stretch of 24 nucleotides of the vtRNA1-1 central domain as being essential for successful maintenance of apoptosis resistance. This study describes a cell signaling-dependent contribution of the human vtRNA1-1 to starvation-induced programmed cell death.

Competition for amino acid flux among translation, growth and detoxification in bacteria.

Transfer-tRNAs (tRNAs) are central entities for translation that deliver amino acids to the ribosome to translate genetic information in an mRNA-template dependent manner. Recent discoveries from our laboratory show that in E. coli and B. licheniformis, some tRNAs are poorly charged despite the plentiful intracellular cognate amino acid. Specifically, tRNAs carrying amino acids that exert toxicity and inhibit bacterial growth when added separately to the growth medium are poorly charged. Here, we discuss various evolutionary strategies different bacterial cells have adopted to precisely hone the competition between amino acid utilization for translation and proliferation and combat the inhibitory effect toward maximizing bacterial fitness. These data add a new twist to the amino acid flux models and to our understanding of the complex intimate link between dynamics of translation and bacterial growth.

Growth-Rate Dependent Regulation of tRNA Level and Charging in Bacillus licheniformis.

Cellular growth crucially depends on protein synthesis and the abundance of translational components. Among them, aminoacyl-tRNAs play a central role in biosynthesis and shape the kinetics of mRNA translation, thus influencing protein production. Here, we used microarray-based approaches to determine the charging levels and tRNA abundance of Bacillus licheniformis. We observed an interesting cross-talk among tRNA expression, charging pattern, and growth rate. For a large subset of tRNAs, we found a co-regulated and augmented expression at high growth rate. Their tRNA aminoacylation level is kept relatively constant through riboswitch-regulated expression of the cognate aminoacyl-tRNA-synthetase (AARS). We show that AARSs with putative riboswitch-controlled expression are those charging tRNAs with amino acids which disfavor cell growth when individually added to the nutrient medium. Our results suggest that the riboswitch-regulated AARS expression in B. licheniformis is a powerful mechanism not only to maintain a constant ratio of aminoacyl-tRNA independent of the growth rate but concomitantly to control the intracellular level of free amino acids.

A new method for long-term storage of titred microbial standard solutions suitable for microbiologic quality control activities of pharmaceutical companies.

Commercially available lyophilized microbial standards are expensive and subject to reduction in cell viability due to freeze-drying stress. Here we introduce an inexpensive and straightforward method for in-house microbial standard preparation and cryoconservation that preserves constant cell titre and cell viability over 14 months.

Quantifying the 'escapers' among RNA species.

tRNAs are fundamental components of translation and emerging evidence places them more centrally in various other cellular processes. However, rather than being uniformly conserved, tRNA abundance is instead highly variable and adaptable. The amount of tRNA genes greatly differs among species. Moreover, even within the same genome, tRNA abundance shapes the proteome in a tissue- and cell-specific manner and is dynamically regulated in response to stress. Here, we review approaches for identification and quantification of tRNAs and their functional integrity. We discuss the resolution of each method and highlight new approaches with cell-wide resolution based on deep-sequencing technologies.