Journal Article

Tunable protein synthesis by transcript isoforms in human cells

Floor, Stephen N, Doudna, Jennifer A

2016

Overview

Eukaryotic genes generate multiple RNA transcript isoforms though alternative transcription, splicing, and polyadenylation. However, the relationship between human transcript diversity and protein production is complex as each isoform can be translated differently. We fractionated a polysome profile and reconstructed transcript isoforms from each fraction, which we term Transcript Isoforms in Polysomes sequencing (TrIP-seq). Analysis of these data revealed regulatory features that control ribosome occupancy and translational output of each transcript isoform. We extracted a panel of 5′ and 3′ untranslated regions that control protein production from an unrelated gene in cells over a 100-fold range. Select 5′ untranslated regions exert robust translational control between cell lines, while 3′ untranslated regions can confer cell type-specific expression. These results expose the large dynamic range of transcript-isoform-specific translational control, identify isoform-specific sequences that control protein output in human cells, and demonstrate that transcript isoform diversity must be considered when relating RNA and protein levels. To produce a protein, a gene’s DNA is first copied to make molecules of messenger RNA (mRNA). The mRNAs pass through a molecular machine known as the ribosome, which translates the genetic code to make a protein. Not all of an mRNA is translated to make a protein; the “untranslated” regions play crucial roles in regulating how much of the protein is produced. In animals, plants and other eukaryotes, many mRNAs are made up of small pieces that are “spliced” together. During this process, proteins are deposited on the mRNA to mark the splice junctions, which are then cleared when the mRNA is translated. Many different mRNAs can be produced from the same gene by splicing different combinations of RNA pieces. Each of these mRNA “isoforms” can, in principle, contain a unique set of features that control its translation. Hence each mRNA isoform can be translated differently so that different amounts of the corresponding protein product are produced. However, the relationship between the variety of isoforms and the control of translation is complex and not well understood. To address these questions, Floor and Doudna measured the translation of over 60,000 mRNA isoforms made from almost 14,000 human genes. The experiments show that untranslated regions at the end of the mRNA (known as the 3′ end) strongly influence translation, even if the protein coding regions remain the same. Furthermore, the data showed that mRNAs with more splice junctions are translated better, implying an mRNA has some sort of memory of how many junctions it had even after the protein markers have been cleared. Next, Floor and Doudna inserted regulatory sequences from differently translated isoforms into an unrelated “reporter” gene. This dramatically changed the amount of protein produced from the reporter gene, in a manner predicted by the earlier experiments. Untranslated regions at the beginning of the mRNAs (known as the 5′ end) controlled the amount of protein produced from the reporter consistently across different types of cells from the body. On the other hand, the 3′ regions can tune the level of protein production in particular types of cells. Floor and Doudna’s findings demonstrate that differences between mRNA isoforms of a gene can have a big effect on the level of protein production. Changes in the types of mRNA made from a gene are often associated with human diseases, and these findings suggest one reason why. Additionally, the ability to engineer translation of an mRNA using the data is likely to aid the development of mRNA-based therapies.

• Publisher: eLife Sciences Publications Ltd,eLife Sciences Publications, Ltd
• Subject: Alternative Splicing / Cell Line / Cell lines / Computational and Systems Biology / Data processing / deep sequencing / Embryos / engineered translation / Epithelial Cells - metabolism / Gene expression / Gene Expression Regulation / Genes and Chromosomes / Genomes / Genomics / Humans / Isoforms / Ontology / Polyadenylation / Polyribosomes / Polyribosomes - metabolism / Protein Biosynthesis / Protein folding / Protein Isoforms - biosynthesis / Proteins / Ribonucleic acid / RNA / RNA processing / RNA, Messenger - metabolism / RNA-seq / Sucrose / transcript isoforms / Transcription / Translation / translational control