PTT-quant: a new method for direct identification and absolute quantification of premature transcription termination events, following the example of bacterial riboswitches.

Regulation of gene expression by premature termination of transcription has been well described in all domains of life, including metazoans, yeast, plants, and bacteria. Although methods for identification of such regulatory events by sequencing are available, the focused biochemical studies of the mechanism are hampered by lack of highly sensitive and accurate experimental methods. Here, we propose a new method for absolute quantification of premature transcription termination events, PTT-quant. It is based on highly sensitive two-step digital droplet PCR protocol, coupled with normalized cDNA synthesis attained by site-specific pre-cleavage of investigated transcripts with RNase H. As a consequence, our method enables the reliable and sensitive quantification of both, prematurely terminated and full-length transcripts. By application of our method to investigation of transcriptional riboswitches in Bacillus subtilis, we were able to precisely measure the dynamics of S-adenosylmethionine (SAM) riboswitch induction, which turned to be ~ 23% higher in comparison the results obtained without cDNA synthesis normalization.Key points• A novel method for quantification of premature transcription termination events was established.• PTT-quant measures absolute concentration of full-length and terminated transcripts.• RNase H and the digital droplet PCR technique is used in PTT-quant.

Regulation of the p53 expression profile by hnRNP K under stress conditions.

The p53 protein is one of the transcription factors responsible for cell cycle regulation and prevention of cancer development. Its expression is regulated at the transcriptional, translational and post-translational levels. Recent years of research have shown that the 5' terminus of p53 mRNA plays an important role in this regulation. This region seems to be a docking platform for proteins involved in p53 expression, particularly under stress conditions. Here, we applied RNA-centric affinity chromatography to search for proteins that bind to the 5' terminus of p53 mRNA and thus may be able to regulate the p53 expression profile. We found heterogeneous nuclear ribonucleoprotein K, hnRNP K, to be one of the top candidates. Binding of hnRNP K to the 5'-terminal region of p53 mRNA was confirmed in vitro. We demonstrated that changes in the hnRNP K level in the cell strongly affected the p53 expression profile under various stress conditions. Downregulation or overexpression of hnRNP K caused a decrease or an increase in the p53 mRNA amount, respectively, pointing to the transcriptional mode of expression regulation. However, when hnRNP K was overexpressed under endoplasmic reticulum stress and the p53 amount has elevated no changes in the p53 mRNA level were detected suggesting translational regulation of p53 expression. Our findings have shown that hnRNP K is not only a mutual partner of p53 in the transcriptional activation of target genes under stress conditions but it also acts as a regulator of p53 expression at the transcriptional and potentially translational levels.

The ample world of riboswitches / Bogaty swiat ryboprzelaczników.

Constantly changing environment requires bacteria to respond quickly and decisively at the cellular level. Notwithstanding, bacteria operate with a limited set of resources restricted to a single cell at their disposal. The natural effect of this state of things for bacteria was a development of a variety of different mechanisms for gene expression control. Among them, riboswitches, defined as sequences present in transcripts with their ability of direct binding low-molecular metabolites and inducing a regulatory response, are considered to be one of the most interesting mechanism of gene regulation. In this publication, there is presented current state of knowledge concerning riboswitches, including such aspects as: mechanisms of action of riboswitches, their structure and diversity as well as their distribution. Furthermore, the history of their discovery at the beginning of the 21st century, and current research that may find future, practical applications.

Transient expression of an adenine base editor corrects the Hutchinson-Gilford progeria syndrome mutation and improves the skin phenotype in mice.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature ageing disorder caused by a point mutation in the LMNA gene (LMNA c.1824 C > T), resulting in the production of a detrimental protein called progerin. Adenine base editors recently emerged with a promising potential for HGPS gene therapy. However adeno-associated viral vector systems currently used in gene editing raise concerns, and the long-term effects of heterogeneous mutation correction in highly proliferative tissues like the skin are unknown. Here we use a non-integrative transient lentiviral vector system, expressing an adenine base editor to correct the HGPS mutation in the skin of HGPS mice. Transient adenine base editor expression corrected the mutation in 20.8-24.1% of the skin cells. Four weeks post delivery, the HGPS skin phenotype was improved and clusters of progerin-negative keratinocytes were detected, indicating that the mutation was corrected in both progenitor and differentiated skin cells. These results demonstrate that transient non-integrative viral vector mediated adenine base editor expression is a plausible approach for future gene-editing therapies.

Levels of sdRNAs in cytoplasm and their association with ribosomes are dependent upon stress conditions but independent from snoRNA expression.

In recent years, a number of small RNA molecules derived from snoRNAs have been observed. Findings concerning the functions of snoRNA-derived small RNAs (sdRNAs) in cells are limited primarily to their involvement in microRNA pathways. However, similar molecules have been observed in Saccharomyces cerevisiae, which is an organism lacking miRNA machinery. Here we examined the subcellular localization of sdRNAs in yeast. Our findings reveal that both sdRNAs and their precursors, snoRNAs, are present in the cytoplasm at levels dependent upon stress conditions. Moreover, both sdRNAs and snoRNAs may interact with translating ribosomes in a stress-dependent manner. Likely consequential to their ribosome association and protein synthesis suppression features, yeast sdRNAs may exert inhibitory activity on translation. Observed levels of sdRNAs and snoRNAs in the cytoplasm and their apparent presence in the ribosomal fractions suggest independent regulation of these molecules by yet unknown factors.

Emerging applications of riboswitches - from antibacterial targets to molecular tools.

The ability to precisely regulate gene expression is one of the most important features of the living cells as it enables the adaptation and survival in different environmental conditions. The majority of regulatory mechanisms involve protein action, however, multiple genes are controlled by nucleic acids. Among RNA-based regulators, the riboswitches present a large group of specific domains within messenger RNAs able to respond to small metabolites, tRNA, secondary messengers, ions, vitamins or amino acids. A simple, accurate, and efficient mechanism of action as well as easiness in handling and engineering make the riboswitches a potent practical tool in industry, medicine, pharmacy or environmental protection. Hereby, we summarize the current achievements and challenges in designing and practical employment of the riboswitch-based tools.

The widespread occurrence of tRNA-derived fragments in Saccharomyces cerevisiae.

Short RNAs derived from the cleavage of tRNA molecules are observed in most organisms. Their occurrence seems to be induced by stress conditions, but still little is known about their biogenesis and functions. We find that the recovery of tRNA fragments depends on the RNA isolation method. Using an optimized RNA extraction protocol and northern blot hybridization technique, we show that the tRNA-derived fragments in yeast are widespread in 12 different growth conditions. We did not observe significant stress-dependent changes in the amounts of tRNA fragments pool. Instead, we show the differential processing of almost all individual tRNAs. We also provide evidence that 3'-part-derived tRNA fragments are as abundant as the 5'- one in Saccharomyces cerevisiae. The resulting set of S. cerevisiae tRNA fragments provides a robust basis for further experimental studies on biological functions of tRFs.