Hin zum molekularen Mechanismus der ranc_RNA18 Funktion: Ist diese ncRNA ein globaler Translationsinhibitor?
Zusammenfassung der Projektergebnisse
Regulation of protein biosynthesis in changing environmental conditions is crucial to maintain cellular homeostasis. Recently, a new class of translational regulators emerged encompassing ribosome-associated non-coding RNAs (rancRNAs) that directly bind to ribosomes allowing a rapid adaptation of the translational process when cells encounter stress situations. In yeast, rancRNA_18, derived from the TRM10 locus, was shown to bind to ribosomes and to be important for rapid shut down of translation and efficient growth resumption under hyperosmotic conditions. However, one question remained to be answered, that is whether rancRNA_18 down regulates protein synthesis on a global scale or whether its binding to ribosomes leads to the generation of “specialized ribosomes” that translate stress-relevant mRNAs. In order to elucidate which scenario is true we electroporated synthetic rancRNA_18 into spheroplasts derived from trm10Δ cells resulting in about 200,000 RNA molecules per cell without any endogenous rancRNA_18 present. As a control, rancRNA_18-M2 was used that contains two synonymous point mutations that render the ncRNA unable to bind to ribosomes and to inhibit translation. Afterwards, the spheroplasts were recovered to allow translation in the presence and absence of rancRNA_18 followed by polysome profiling and isolation of actively translated mRNAs from polysomal fractions that were eventually sequenced. Evaluation of the obtained sequencing data revealed that there are no mRNAs that are preferentially translated in the presence of rancRNA_18. Thus, these results suggest that rancRNA_18 is a translational inhibitor that rapidly attenuates protein synthesis on a global scale to open a time window for adaptation processes to be established in case of high salinity. These finding prompted us to refrain from performing ribosome profiling as initially planned. Importantly, the results obtained from RNA sequencing contribute to a publication giving insight into the function and processing of rancRNA_18 that is currently in preparation.
