High levels of tRNA abundance and alteration of tRNA charging by bortezomib in multiple myeloma

Abstract

In multiple myeloma (MM), malignant plasma cells produce large amounts of antibodies and have highly active protein translational machinery. It is not known whether regulation of the abundance and aminoacylation (charging) of transfer RNA (tRNA) takes place in myeloma cells to accommodate for the increased amount of protein translation. Using tRNA-specific microarrays, we demonstrate that tRNA levels are significantly elevated in MM cell lines compared to normal bone marrow cells. We furthermore show that the addition of the proteasome inhibitor, bortezomib (Velcade, PS-341) results in decreased charging levels of tRNAs, in particular those coding for hydrophobic amino acids. These results suggest that tRNA properties are altered in MM to accommodate for its increased need for protein translation, and that proteasome inhibition directly impacts protein synthesis in MM through effects on tRNA charging.

Fig. 1. Schemes for measuring tRNA abundance (A) and aminoacylation (charging, B)

(A) Total RNA was deacylated to remove 3' amino acids. An RNA-DNA hybrid oligo containing Cy3 or Cy5 was ligated to the 3' end of tRNA using T4 DNA ligase. Fluorescent-labeled tRNAs were directly hybridized to microarrays containing tRNA-specific probes. (B) Total charged tRNA sample was split in two halves. One half was treated with periodate which oxidized only uncharged tRNAs and represents the amount of charged tRNA; the other half was not treated with periodate but otherwise treated identically and represented the amount of total tRNA. After removal of periodate, tRNAs were deacylated and labeled with the Cy3/Cy5 containing RNA-DNA hybrid oligos.

Fig. 2. Comparing tRNA abundance in MM cell lines and normal bone marrow

(A) Mean and Median values of chromosomal-encoded and mitochondrial-encoded tRNAs in MM lines compared to bone marrow cells. (B) Ratio of each probe for the sample with lowest (bone marrow) and highest (H929) relative tRNA abundance, plotted against the sample with intermediate tRNA abundance (U266). Left: chromosomal-encoded tRNAs; right: mitochondrial-encoded tRNAs. (C) Relative abundance of MM.1R (dexamethasone-resistent) versus MM.1S (dexamethasone-sensitive) showing similar tRNA levels in these two lines derived from the same parental line. Chromosomal-encoded tRNAs are in black and mitochondrial-encoded tRNAs are in red. tRNA^Ile(TAT) shows highest deviation between these two lines (1.7-fold). (D) Relative abundance of tRNA isoacceptor families after normalization to the Median value of all chromosomal-encoded tRNAs in the respective samples. Only isoacceptor families are shown that can be distinguished by our tRNA microarray and have the potential for translational regulation. The significant difference among arginine, leucine isoacceptor families (arrows) suggests the possibility for adjusting translational efficiency in genes with distinct codon usages. The relative abundance of initiator-tRNA^Met (tRNA-i) after normalization to the Median value is also shown.

Fig. 3. Comparing tRNA charging levels in MM cell lines upon Bortezomib treatment

(A) H929 line before Bortezomib addition (0h). “Charging level” represents fluorescent ratios of periodate-treated tRNA/total tRNA. (B) H929 line at 0h and 2h after Bortezomib addition. A small, identical section of the microarray image is shown. Total tRNA is in green and charged tRNA is in red. More green spots are present in the 2h sample, indicating decreased tRNA charging levels. Filled squares in the grid indicate human tRNA probes and are expected to show fluorescent signals. Open squares in the grid indicate non-human tRNA probes and are expected to be devoid of fluorescent signals. (C) TreeView presentation of changes in tRNA charging levels, grouped by amino acid properties. Chromosomal-encoded tRNAs are above and mitochondrial-encoded tRNAs are below the dashed line. Gray columns divide the cell lines and gray rows divide tRNAs grouped by amino acid properties. Charging level changes are normalized to the 0h sample of each respective cell line. The dominant feature is the decrease in charging of chromosomal-encoded tRNAs coding for hydrophobic amino acids, only in the Bortezomib sensitive lines at 2h (H929 and MM.1S), but not in the resistant line (RPMI8266). This feature is maintained at higher concentration of Bortezomib for MM1S line (MM.1S*).