Organ-specific translation elongation rates measured by in vivo ribosome profiling

Abstract

Protein synthesis and degradation are intricate biological processes involving more than a hundred proteins operating in a highly orchestrated fashion. Despite the progress, few options are available to access translation in live animals as the increase in animal's complexity limits the repertoire of experimental tools that could be applied to observe and manipulate processes within animal's body, organs, and individual cells. It this study, we developed a labeling-free method for measuring organ-and cell-type specific translation elongation rates. It is based on a time-resolved delivery of translation initiation and elongation inhibitors in live animals followed by ribosome profiling. It also reports translation initiation sites in an organ-specific manner. Using this method, we found that the elongation rates differ among mouse organs and determined them to be 6.8, 5.2, and 4.4 amino acids per sec for liver, kidney, and skeletal muscle, respectively. protein synthesis | translation | elongation | ribosome profiling | mouse P rotein synthesis is regulated both at the transcriptional (availability of mRNA) and translational (the number of active ribosomes, translational efficiency, the speed of elongation and the frequency of initiation events) levels. This complexity presents endless possibilities for malfunctioning. In turn, the vast majority of cellular functions are carried by proteins, making any aberrations in their biosynthesis potentially harmful. Modern in vitro methods allow unsurpassed precision and flexibility, e.g. real-time translation dynamics of a single molecule can be monitored with fluorescent microscopy (1, 2), and transcriptome-wide snapshots of translation with a single nucleotide resolution can be achieved by means of ribosome profiling (3-6). In contrast to research performed in cells, few options are currently available to investigate translation in live animals. It is particularly challenging to assay specific steps of translation, i.e. elongation, initiation or termination rate, in vivo. Several clever approaches were designed, including animals fed with amino acid supplements labeled with a stable isotope (7) or radioactively labeled amino acids. To estimate the average translation elongation speed, short-term pulses of radioactively labeled amino acids can be injected directly into the portal vein of an animal (8). The radiation accumulation rate can be used to project the average translation elongation rate (8,9); however, tissue samples are rendered by this method unsuitable for many other molecular biology assays.We were guided by these challenges and the need to assess various steps of translation in various organs of live animals at an individual gene level. In this work, we present a technique to directly assess translation in vivo and measure organ-and cell-specific translation elongation rates and other features of protein synthesis. Our method requires no radioactive labeling or transgenic reporters and can be applied to a variety of small animals such as a mouse or a rat. It...

Genetic removal of p70 S6K1 corrects coding sequence length-dependent alterations in mRNA translation in fragile X syndrome mice

“…2E). HHT inhibits translation initiation rapidly (Gerashchenko et al, 2018). Therefore, after its addition, puromycin labeling specifically measures the elongation phase of protein synthesis.…”

Multi-faceted deregulation of gene expression and protein synthesis with age

“…Tissue samples (~55 mg and ~75 mg for liver and kidney, respectively) were used for subsequent analyses. Tissues were lysed as described previously (4). After homogenization and centrifugation, 250 µl of lysate were supplied with 20 Units of SUPERase-In RNase inhibitor and taken for RNA-Seq library preparation, and the 500 µl of lysate was brought to 1 ml with lysis buffer and taken for Ribo-Seq library preparation.…”

“…Ribosome profiling libraries were prepared as described previously (4) with the modification in RNase digestion as indicated below. RNA digestion of lysates was performed for 1 hour with the mixture of 2000 Units of RNase T1 (Epicentre) and 300 Units of RNase S7 (Roche/Sigma).…”

Accumulation of “Old Proteins” and the Critical Need for MS‐based Protein Turnover Measurements in Aging and Longevity

“…Aside from fluorescent and chemical labeling approaches, a method of directly assessing translational elongation rates in live animal in vivo has been very recently developed. This method applies the time‐resolved delivery of specific inhibitors of translational initiation and elongation in combination with polysome profiling to determine rates of translational elongation, both as tissue averages and at the level of individual transcripts …”

“…This method applies the time-resolved delivery of specific inhibitors of translational initiation and elongation in combination with polysome profiling to determine rates of translational elongation, both as tissue averages and at the level of individual transcripts. [53] Alternative MS-based workflows have also been employed to identify and quantify the relative abundances or PTM-status of known, or presumed, LLPs. Protein aggregates and insoluble inclusions, for example, tend to be long-lived because they evade protein turnover machinery.…”