RNase T1 is the best known representative of a large family of ribonucleolytic proteins secreted by fungi, mostly Aspergillus and Penicillium specie

Fungi establish a complex network of biological interactions with other organisms in nature. In many cases, these involve the production of toxins for survival or colonization purposes. Among these toxins, ribotoxins stand out as promising candidates for their use in biotechnological applications. They constitute a group of highly specific extracellular ribonucleases that target a universally conserved sequence of RNA in the ribosome, the sarcin-ricin loop. The detailed molecular study of this family of toxic proteins over the past decades has highlighted their potential in applied research. Remarkable examples would be the recent studies in the field of cancer research with promising results involving ribotoxin-based immunotoxins. On the other hand, some ribotoxin-producer fungi have already been studied in the control of insect pests. The recent role of ribotoxins as insecticides could allow their employment in formulas and even as baculovirus-based biopesticides. Moreover, considering the important role of their target in the ribosome, they can be used as tools to study how ribosome biogenesis is regulated and, eventually, may contribute to a better understanding of some ribosomopathies.

Ribotoxins are potent inhibitors of protein biosynthesis and inactivate ribosomes from a variety of organisms. The ribotoxin α-sarcin cleaves the large 23S ribosomal RNA (rRNA) at the universally conserved sarcin–ricin loop (SRL) leading to complete inactivation of the ribosome and cellular death. The SRL interacts with translation factors that hydrolyze GTP, and it is important for their binding to the ribosome, but its precise role is not yet understood. We studied the effect of α-sarcin on defined steps of translation by the bacterial ribosome. α-Sarcin-treated ribosomes showed no defects in mRNA and tRNA binding, peptide-bond formation and sparsomycin-dependent translocation. Cleavage of SRL slightly affected binding of elongation factor Tu ternary complex (EF-Tu•GTP•tRNA) to the ribosome. In contrast, the activity of elongation factor G (EF-G) was strongly impaired in α-sarcin-treated ribosomes. Importantly, cleavage of SRL inhibited EF-G binding, and consequently GTP hydrolysis and mRNA–tRNA translocation. These results suggest that the SRL is more critical in EF-G than ternary complex binding to the ribosome implicating different requirements in this region of the ribosome during protein elongation.

Ribotoxins are a family of highly specific fungal ribonucleases that inactivate the ribosomes by hydrolysis of a single phosphodiester bond of the 28 S rRNA. alpha-Sarcin, the best characterized member of this family, is a potent cytotoxin that promotes apoptosis of human tumor cells after internalization via endocytosis. This latter ability is related to its interaction with phospholipid bilayers. These proteins share a common structural core with nontoxic ribonucleases of the RNase T1 family. However, significant structural differences between these two groups of proteins are related to the presence of a long amino-terminal beta-hairpin in ribotoxins and to the different length of their unstructured loops. The amino-terminal deletion mutant Delta(7-22) of alpha-sarcin has been produced in Escherichia coli and purified to homogeneity. It retains the same conformation as the wild-type protein as ascertained by complete spectroscopic characterization based on circular dichroism, fluorescence, and NMR techniques. This mutant exhibits ribonuclease activity against naked rRNA and synthetic substrates but lacks the specific ability of the wild-type protein to degrade rRNA in intact ribosomes. The results indicate that alpha-sarcin interacts with the ribosome at two regions, i.e. the well known sarcin-ricin loop of the rRNA and a different region recognized by the beta-hairpin of the protein. In addition, this latter protein portion is involved in interaction with cell membranes. The mutant displays decreased interaction with lipid vesicles and shows behavior compatible with the absence of one vesicle-interacting region. In agreement with this conclusion, the deletion mutant exhibits a very low cytotoxicity on human rhabdomyosarcoma cells.