Alternative splicing of MEFV in mouse spleen cells




Marisa Vomvos

Introduction

The MEFV gene is located on the short arm of chromosome 16. It contains 10 exons and encodes a protein called pyrin. Pyrin is primarily expressed in certain white blood cells, such as monocytes, granulocytes, and dendritic cells, as well as in synovial fibroblasts. Pyrin's main role is in regulating innate immunity (Chae, 2009). Pyrin has both pro- and anti-inflammatory roles, which impact the interleukin-1 activation pathway (Manukyan, 2016).

Mutations in MEFV that affect pyrin function are responsible for an auto inflammatory disorder called Familial Mediterranean fever (FMF). This disorder is one of the most frequent genetic disorders in Mediterranean populations, including Turkish, Arab, Armenian, and Jewish (Jéru, 2013). In these at-risk populations, FMF has a frequency ranging from 1 in 200 to 1 in 1,000.

FMF is characterized by recurrent episodes of fever and painful inflammation attacks in the chest, joints, and/or abdomen, which may last hours to a few days (Jéru, 2013). The characteristics and severity of the disorder vary from patient to patient, namely due to the specific mutation they inherit (Touitou, 2001). This variation can be in how long the attack lasts, how frequent the patient has them, and which body part the inflammation is localized to. Between attacks, individuals with FMF are asymptomatic.

Although FMF is inherited in an autosomal recessive manner, there are often cases of heterozygotes with only one mutated allele showing clinical symptoms of FMF. Heterozygous individuals actually account for up to 30% of FMF patients (Jeru, 2013). The idea for this study stemmed from these cases, namely that environmental cues could be triggering episodes by reducing the amount of functional pyrin produced by the one wild-type allele. The purpose of this study was to analyze the impact of various compounds on MEFV expression, and to investigate any alternatively spliced transcript variants produced.


Materials and Methods

Cells were harvested from mice spleens, which were removed from sacrificed mice. The cells were then treated with various agents for 24 hours. Untreated cells were used as a control.

After 24 hours, RNA was isolated from the cells as follows. Cells were transferred to 1.5mL Eppendorf tubes, and centrifuged to pellet. Media was removed off the top, and the pellet was resuspended in RLT lysis buffer plus B-mercaptoethanol. A Qiagen Qia-Shredder column was used to remove genomic DNA. RNA was purified using the Qiagen RNeasy mini kit.

RT-PCR analysis was performed on the RNA samples utilizing the Qiagen OneStep RT-PCR kit. Two different primer pairs were used: the first spanned exon 1 to exon 8, while the second spanned exon 2 to exon 3.

PCR products were visualized by carrying out gel electrophoresis on a 1% agarose gel with ethidium bromide.

PCR products were also purified following QIAquick RNA purification protocol, and sent to GeneWiz for sequencing.

Band intensities were quantified utilizing ImageJ software, and a ratio of upper band to lower band intensities was calculated for each sample.


Results

The first amplification used a primer pair spanning exon 1 to exon 8 (Figure 1). The results show that two MEFV transcript variants are being expressed in the cells. Sequencing analysis of the purified PCR products determined that the two variants differ by 90 bases. The shorter transcript matched the expected sequence for MEFV, while the longer transcript contained the same sequence, but also included the first 90bp of intron 2 (Figure 2).

The amplification in Figure 3 used a primer pair spanning exon 2 to exon 3 (primer pair shown in Figure 2). The two transcript variants were present in all of the cells. In Figure 3, numbers at the top of the lanes correspond to compounds used to treat the cells, while numbers at the bottom of the lanes correspond to ratios of upper to lower band intensities, calculated as follows:

ImageJ software was used to quantify the intensities of the upper and lower bands of each sample. Then, a ratio of the upper band (+90bp transcript) divided by the lower band was calculated. While most of the samples had a ratio within the range of 1.18 – 1.38, compound 32 had a ratio of 1.87 and compound 205 had a ratio of 2.71 (Figure 3).


Discussion

In this study, it was found that two MEFV transcript variants are produced in mouse spleen cells. Upon sequencing analysis, it was determined that these transcripts differ by 90 bases in exon 2. The longer transcript has an extra 90 bases, which are spliced into the transcript from intron 2. This +90bp transcript is likely produced due to a sequence within intron 2 (GTTAGT), which is nearly identical to the donor splice site consensus sequence (GTRAGT). Exon 2 codes for a region in the protein’s N-terminus, which has been shown to function in microtubule binding, as well as localization to the nucleus when pyrin is cleaved by caspase 1. The +90bp transcript remains in frame, and will introduce an additional 30 amino acids into this region of the protein. If this inclusion has an effect on protein function, it may have implications in FMF.

The results suggest that treatment with certain agents can manipulate alternative splicing of these MEFV transcripts. The bands corresponding to the RT-PCR products from untreated cells in Figure 3 appear to be approximately equal in intensity, suggesting that there are relatively equal amounts of the two transcript variants present in these cells. To determine whether or not the various agents used to treat the other cell samples impacted the relative prevalence of these transcripts, ImageJ software was used to quantify a ratio of the upper band (+90bp transcript) intensity divided by the lower band intensity. While most of the samples’ ratios range from 1.18 – 1.38, compound 32 reached 1.87 and compound 205 reached 2.71 (Figure 3). This demonstrates an increase in the relative prevalence of the +90bp transcript in these cells compared to the shorter transcript.

Further studies should be done on the protein level to better understand the biological significance of the two MEFV isoforms.

References

1. Chae JJ, Aksentijevich I, Kastner DL. (2009). Advances in the understanding of Familial Mediterranean fever and possibilities for targeted therapy. British Journal of Haemotology, 146(5), 467-478.
2. Jéru I, Hentgen V, Cochet E, Duquesnoy P, Le Borgne G, et al. (2013). The Risk of Familial Mediterranean Fever in MEFV Heterozygotes: A Statistical Approach. PLoS ONE 8(7): e68431
3. Manukyan, G, & Aminov, R. (2016). Update on Pyrin Functions and Mechanisms of Familial Mediterranean Fever. Frontiers in Microbiology, 7, 456.
4. Touitou, I. (2001). The spectrum of Familial Mediterranean Fever (FMF) mutations. European Journal of Human Genetics, 9(7), 473-483.

Figures


Figure 1-RT-PCR Results Show Two Transcript Variants. Primer pair spanned exon 1 to exon 8, and products were visualized on 1% agarose gel with EtBr.


Figure 2-The two MEFV transcript variants based on sequencing analysis. Arrows represent primer pair used in subsequent RT-PCR reaction.


Figure 3-RT-PCR Results and Relative Band Intensities. Primer pair spanned exon 2 to exon 3, and products were visualized on 1% agarose gel with EtBr.


The MEFV gene encodes a protein called pyrin. Pyrin has several roles involved in regulating innate immunity. Mutations in the MEFV gene cause an auto inflammatory disorder known as Familial Mediterranean fever (FMF). The purpose of this study is to analyze the impact of various compounds on MEFV expression, and to investigate any alternatively spliced transcript variants that are produced. Mouse spleen cells were treated with various compounds, and RNA samples isolated from the cells were used for RT-PCR analysis. Two transcript variants were found to be present in the cells, and sequencing analysis determined one transcript has an additional 90 bases spliced into exon 2 from intron 2. Two compounds were found to increase the relative prevalence of the +90bp variant. Future studies should be done on the protein level to understand the isoforms’ biological significance.

Full Paper

Acknowledgments

I would like to thank Dr. Berish Rubin for making this project possible, and for all of his support and advice along the way. I would also like to thank Catharina Grubaugh and Anthony Evans for all of their time, patience, and dedication.


This document was last modified 05/17/2017.
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