The impact of temperature-dependent alternative splicing on protein expression of the helicase eIF4A2

Abstract

Alternative splicing is a co-transcriptional process in which multiple mature mRNAs isoforms can arise from a single pre-mRNA transcript. The translation of these different mRNAs significantly increases the size of the proteome. At the genomic level, alternative splicing leads to the regulation of gene expression as part of RNA processing. Alternative splicing allows the cell to respond quickly to changes in its environment, as it is a rapid process. Changes in a cell's environment are caused, for example, by toxins, infections or temperature variations. Since temperature fluctuations are a ubiquitous factor, further investigation of their effects on splicing, as well as gene and protein expression, was of interest. Homoiothermic organisms such as humans can maintain a constant body temperature, which makes them robust to more severe temperature fluctuations. In contrast, plants and poikilothermic animals are more affected by their ambient temperature. To better understand the impact of temperature on global cellular processes, this work is interested in the effects of temperature-dependent alternative splicing on the protein expression of human helicases. In our study we identified a number of helicases whose expression was affected by changes in temperature. As the DEAD-box RNA helicase eIF4A2 turned out to be the most interesting of the identified proteins, we put our main focus on its investigation. We showed that two temperature-dependent alternative exons regulate its protein expression. While exon 10A was the interest of several previous studies, exon 4 has been neglected in the past. Thus, we examined exon 4 and exon 10A individually and also the co-regulation of the two alternative exons. Therefore, we combined our initial in vitro approach with a specially established CRISPR/Cas9 based cell culture system. Both setups revealed that the inclusion of the alternative exon 4 is causative for a higher mRNA abundance, while the isoform containing exon 10A leads to mRNA degradation via the NMD pathway due to the inclusion of a premature stop codon. Analysis of RNA-seq data and minigenes showed that the two alternative exons are co-regulated despite their spatial distance. A comprehensive analysis of the proteome showed that the absence of exon 4 leads to a complete loss of protein (knockout), whereas exon 4 significantly increases the formation of the full-length protein (constitutive overexpression). As an RNA helicase, the eukaryotic translation initiation factor eIF4A2 is involved in many cellular processes. We have shown that the ratio between eIF4A2 exon 4 and exon 10A is not only temperature dependent but also cell-type specific and that this ratio is regulated by their co-regulated alternative splicing. This tissue-specific expression is highly conserved in many species, raising questions about the exact function eIF4A2. Helicases are essential proteins in cellular metabolism and are expressed in many species. Combined with the effect of temperature, eIF4A2 represents a fascinating RNA helicase that gives global insights into the regulation of helicases in a temperature-dependent manner. Global proteome analysis revealed many proteins of the respiratory chain complex I as direct targets of eIF4A2. Therefore, we propose a role of eIF4A2 in the electron transfer within the respiratory chain in mitochondria via regulating the expression of essential complex I components. Furthermore, our data are consistent with previous studies that showed eIF4A2 functions in neuronal development. In summary, this work provides new insights into regulation of protein expression by alternative splicing in response to temperature using the eukaryotic RNA helicase eIF4A2 as an example.