No abstract

For efficient aminoacylation, tRNAs carry the conserved 3′-terminal sequence C-C-A, which is synthesized by highly specific tRNA nucleotidyltransferases (CCA-adding enzymes). In several prokaryotes, this function is accomplished by separate enzymes for CC- and A-addition. As A-adding enzymes carry an N-terminal catalytic core identical to that of CCA-adding enzymes, it is unclear why their activity is restricted. Here, it is shown that C-terminal deletion variants of A-adding enzymes acquire full and precise CCA-incorporating activity. The deleted region seems to be responsible for tRNA primer selection, restricting the enzyme’s specificity to tRNAs ending with CC. The data suggest that A-adding enzymes carry an intrinsic CCA-adding activity that can be reactivated by the introduction of deletions in the C-terminal domain. Furthermore, a unique subtype of CCA-adding enzymes could be identified that evolved out of A-adding enzymes, suggesting that mutations and deletions in nucleotidyltransferases can lead to altered and even more complex activities, as a simple A-incorporation is converted into sequence-specific addition of C and A residues. Such activity-modifying events may have had an important role in the evolution of tRNA nucleotidyltransferases.

Transfer RNAs (tRNAs) require the absolutely conserved sequence motif at their 3′-ends, representing the site of aminoacylation. In the majority of organisms, this trinucleotide sequence is not encoded in the genome and thus has to be added post-transcriptionally by the CCA-adding enzyme, a specialized nucleotidyltransferase. In eukaryotic genomes this ubiquitous and highly conserved enzyme family is usually represented by a single gene copy. Analysis of published sequence data allows us to pin down the unusual evolution of eukaryotic CCA-adding enzymes. We show that the CCA-adding enzymes of animals originated from a horizontal gene transfer event in the stem lineage of Holozoa, i.e. Metazoa (animals) and their unicellular relatives, the Choanozoa. The tRNA nucleotidyltransferase, acquired from an α-proteobacterium, replaced the ancestral enzyme in Metazoa. However, in Choanoflagellata, the group of Choanozoa that is closest to Metazoa, both the ancestral and the horizontally transferred CCA-adding enzymes have survived. Furthermore, our data refute a mitochondrial origin of the animal tRNA nucleotidyltransferases.

The underlying molecular mechanisms of the aberrant expression of components of the HLA class I antigen processing and presentation machinery (APM) in tumors leading to evasion from T cell-mediated immune surveillance could be due to posttranscriptional regulation mediated by microRNAs (miRs). So far, some miRs controlling the expression of different APM components have been identified. Using in silico analysis and an miR enrichment protocol in combination with small RNA sequencing, miR-26b-5p and miR-21-3p were postulated to target the 3′ untranslated region (UTR) of the peptide transporter TAP1, which was confirmed by high free binding energy and dual luciferase reporter assays. Overexpression of miR-26b-5p and miR-21-3p in melanoma cells downregulated the TAP1 protein and reduced expression of HLA class I cell surface antigens, which could be reverted by miR inhibitors. Moreover, miR-26b-5p overexpression induced a decreased T cell recognition. Furthermore, an inverse expression of miR-26b-5p and miR-21-3p with TAP1 was found in primary melanoma lesions, which was linked with the frequency of CD8+ T cell infiltration. Thus, miR-26-5p and miR-21-3p are involved in the HLA class I-mediated immune escape and might be used as biomarkers or therapeutic targets for HLA class Ilow melanoma cells.

Epithelial-to-mesenchymal transition (EMT) is a crucial step in cancer progression and the number one reason for poor prognosis and worse overall survival of patients. Although this essential process has been widely studied in many solid tumors as e.g. melanoma and breast cancer, more detailed research in renal cell carcinoma (RCC) is required, especially for the major EMT-inducer transforming growth factor beta (TGF-β). Here, we provide a study of six different RCC cell lines of two different RCC subtypes and their response to recombinant TGF-β1 treatment. We established a model system shifting the cells to a mesenchymal cell type without losing their mesenchymal character even in the absence of the external stimulus. This model system forms a solid basis for future studies of the EMT process in RCCs to better understand the molecular basis of this process responsible for cancer progression.