Interferon-gamma Induced 5’ Alternative Splicing of Tryptophanyl-tRNA Synthetase (WaRS)




Shaili Shah

Introduction

Viruses are small obligate parasites that invade the body, and use the host’s machinery to replicate and invade other cells. In response to a virus, the cell stimulates production of interferons (IFNs) which bind to high affinity cell surface receptors and activate the expression of downstream target genes (Lewis et al. 1989). IFNs are part of the non-specific immune system and lead to the induction of an "antiviral state" in cells, which is characterized by inhibition of both viral replication and cell proliferation, as well as the increased ability of natural killer cells to lyse infected cells (Pottathil et al. 1980). The first two interferons identified were IFN-alpha and IFN-beta, which encompasses a family of structurally related cytokines called type I IFNs (Pestka et al. 1987). IFN-gamma, the only type II IFN, initiates signaling by binding to a distinct receptor at the cell surface (Platanias 2005). IFN-gamma is made by T-lymphocytes and natural killer cells, and provides the specific signaling that activates cell based defenses. IFN-gamma triggers production of major histocompatibility complex molecules and changes the composition of proteosomes, so abnormal proteins can be targeted for ubiquitination (Goodsell 2001). Induction of nitric oxide by macrophages via the interferon signaling pathway ensures that the amplified amount of protein aggregations does not lead to apoptosis (Goodsell 2001).

One of the downstream genes induced by IFN-gamma is tryptophanyl-tRNA synthetase (WaRS). The aminoacyl-tRNA synthetases (ARS) are essential enzymes that covalently link amino acids to their cognate tRNAs (Schimmel 1987). The 20 ARS are divided into two groups of ten each; class I enzymes share a common active site architecture which includes a specific type of folded nucleotide-binding domain while class II enzymes are made up of a seven-stranded antiparallel beta-sheet surrounded by alpha-helices. In reactions catalyzed by the class I ARS, the aminoacyl group is coupled to the 2'-hydroxyl of the tRNA. This differs from class II reactions where the 3'-hydroxyl sites are used (Cusack et al. 1990). These two variants in structure for class I and class II synthetases are thought to aid in specificity and sequence adaptions, and might contribute to the additional cellular functions that these enzymes catalyze (Ruff et al. 1991). Alternative splicing of aminoacyl tRNA synthetases occurs in the lysyl-tRNA synthetase (Targoff et al.1993), tyrosyl-tRNA synthetase (Wakasugi et al. 2002), isoleucyl-tRNA synthetase (Nichols et al. 1995), cysteinyl-tRNA synthetase (Kim et al. 2000), and tryptophanyl-tRNA synthetase (Wakasugi and Schimmel 1999). The alternative splice variants might be responsible for the housekeeping functions of the ARS (Schimmel 1987). The fragments of WaRS have anti-angiogenic activity, which provides a biological rationale for their production (Otani et al. 2002). Here I examine the induction of WaRS in response to interferon-gamma and the alternative splice variants produced.

Figures


Figure 1-Primer set A produces three different transcripts; with all the exons included, the transcript would be 319bp, with exon 1b missing it would be 296bp and with exon 2 sliced out the transcript would be182 bp. Primer set B produces a 215bp product.


Figure 2-RT–PCR analysis of IFN-gamma up-regulated WaRS in HeLa cells. Cells were treated with (lanes marked-T) or without (lanes marked- U) IFN-gamma (500 U/ml) for 8 hours. NTC lanes are no template controls. The reaction was done in duplicate or triplicate to avoid errors, to increase product for sequencing and to ensure reproducibility. Total RNAs were isolated and converted to cDNA using RT-PCR as described in Materials and Methods and analyzed by 1% agarose gel electrophoresis. Under these conditions, the differences between the levels of WaRS mRNA could be seen. GAPDH is an internal control.


Figure 3-Alignment of sequencing data using BLAST. 319bp (fig 3a), 296bp (fig 3b), and 182 (fig 3c) product produced from primer set A, Forward –exon 1a and Reverse-exon3. Product produced from primer set B (fig 3d), Forward-exon1b to Reverse-exon1b. The data I generated is in bold in comparison to the GenBank accession sequence (number BC095453) that is underneath it.


Alternative splicing enables the cell to use the same gene to target different proteins, have greater stability, or signal different pathways (Kim et al. 2000). The full length mRNA for WaRS is critical in charging tryptophans and adding them onto tRNAs (Liu et al 2004). My results show that IFN-gamma is a strong stimulator of tryptophanyl-tRNA synthetase. I expected to see an increase in the specific type of alternative splice variant produced, however my results show that after treatment, the proportion of mRNA variants increase uniformly. Instead of a specific variant being up regulated, there is a twofold increase in all the mRNA variants. Contrary to this study, others (Jorgensen et al. 2000, Shaw et al. 1994 and Wakasugi et al. 2002) have shown that the relative amount of each variant is capricious, and it was problematic to quantify the splice variants produced. The quantification may have been difficult if the splice variants for the mRNA was only slightly up regulated, or if the fidelity of Taq polymerase was not uniform across all samples. The differences between the variants was difficult to quantify, however, further work using Real Time PCR should help determine if there is any diversity in the splice variants produced.

The truncated variants produced are cell and situation specific, and have other functions; the shortest transcript has been shown to be essential for activation of angiogenesis activity (Wakasugi et al. 2002), allowing increased blood flow. Due to IFN-gamma ability to stimulate angiogenic cytokines, cleavage of WaRS transcripts might be a type of signaling mechanism. Others (Shaw et al. 1994, Wakasugi et al. 2002) have shown that the shortest 182bp mRNA is specifically responsible for the production of angiogenic cytokines. There is no known role for the 296bp size transcript produced, and further work focused on determining its own function should prove valuable for understanding the role of alternative splicing of WaRS. Further work is needed to determine whether the 182bp truncated transcript with exon 2 missing acts in the same signaling mechanism as other factors responsible for angiogenesis.

Full Paper

Acknowledgments

I would like to thank Jinsong Qiu for his long hours, infinite amounts of wisdom and patience. I would also like to thank Lisa Sarran for her help, Dr. Rubin for his advice, Dr. Henriksen for her assistance through a few sticky situations and my classmates for all their entertaining virtues.


This document was last modified 05/18/2006.
This site is powered by the versatile Zope platform.
This is a project of the Biology Department of Fordham University
Biotechniques.org Home