RNase activity requires formation of disulfide bonds and is regulated by the redox state

Abstract

The activity of many RNases requires the formation of one or more disulfide bonds which can contribute to their stability. In this study, we show that RNase activity and, to a much lesser extent, nuclease activity, are redox regulated. Intracellular RNase activity was altered in vitro by changes in the glutathione redox state. Moreover, RNase activity was abolished following exposure to reducing agents such as beta-ME or DTT. Following reduction with glutathione (GSH), RNase activity could be fully reactivated with oxidized glutathione (GSSG). In contrast, RNase activity could not be reactivated when reduced with DTT. Decreasing the level of glutathione in vivo in wheat increased RNase activity. Tobacco engineered to have an increased glutathione redox state exhibited substantially lower RNase activity during dark-induced senescence. These results suggest that RNase activity requires the presence of one or more disulfide bonds that are regulated by glutathione and demonstrate for the first time that RNase activity can be altered with an alteration in cellular redox state.

DeLTa-Seq: direct-lysate targeted RNA-Seq from crude tissue lysate

“…In this study, we developed Direct-RT buffer that realizes cost-effective direct-lysate RT of crude plant, yeast and animal samples. DTT could reduce disulfide bonds required for the activity of RNase in lysates 24 . Because no loss of RNA in purification occurs through direct-lysate RT, this technique is beneficial also for gene expression analysis in small tissues from which it is difficult to purify enough amount of RNA for the experiments.…”

“…In the previous research 24 , inactivation of RNase in an extract prepared from dark grown wheat required 0.5 mM DTT or more than 0.014% 2-mercaptoethanol (2ME) 24 .…”

“…Although several studies showed that RT can be possible directly from lysate of cultured animal cells in cell-lysis buffers [21][22][23] , in our knowledge, no study showed direct-lysate RT from crude tissue lysates. A previous study showed that reducing regents such as dithiothreitol (DTT) irreversibly deactivate intrinsic RNase in wheat 24 . Yet, whether DTT can portect RNA in the lysate of other speiseses and whether DTT does not inhibit reverse transcription has not been examined.…”

Simultaneous Isolation of mRNA and Native Protein From Minute Samples Of Cells

“…This strategy has previously been used to inhibit the degradation of RNA (2, 3), either by disulfide bridge reduction in RNases (4) or activation of endogenous RNase inhibitors (5). The new protocol was developed using neonatal rat ovaries, human granulosa cells (hGCs), and human corpus luteum (CL) by measuring the mRNA expression of the inhibin alpha subunit ( Inha) gene along with two housekeeping genes, the 60S ribosomal protein L32 (Rpl32) and glyceraldehyde 3-phosphate dehydrogenase (Gapdh), while measuring the enzymatic activities of phosphodiesterases (PDE), caspase-3, lactate dehydrogenase (LDH) and the immunoprecipitation of GAPDH and -actin.…”

Identificación y localización subcelular de proteasas asociadas a la senescencia foliar

“…La actividad ADNasa y ARNasa de la nucleasa bifuncional de Arabidopsis BFN1 aumenta durante la senescencia foliar (Perez-Amador et al, 2000). En general, la actividad nucleasa in vitro es dependiente del estado redox y es inhibida por agentes quelantes (Chen et al, 2004).…”