Alternative splicing of SRSF3 in the THP1 cell line




Fu, Xia

Introduction

SR (Serine/Arginine) proteins are a conserved family of proteins involved in RNA splicing. SR proteins are composed of two domains, the RNA recognition motif region and SR rich domain. Serine/Arginine-rich splicing factor 3(SRSF3) is the smallest member of SR protein family. SRSF3 protein contains one N-terminal RNA-binding domain and a downstream RS-rich domain. It has two transcript variants. Transcript variant 1 can code for 164 amino acids in length with one RRM domain followed by RS region. The transcript variant 2 has a 456 base pairs insertion. And a stop codon in this insertion could shorten the stop coding very early. In general, SRSF3 protein constitutes part of the spliceosome, which is critical for mRNA splicing. Also SRSF3 is a shuffling protein so that it has been shown to be involved in mRNA nuclear export such as of histone H2A and in translation.

The nutraceutical we used in this project was extracted from blessed thistle. Blessed thistle is a plant that has been a part of medicine for its ability to increase appetite and support the digestive process. The goal of this experiment was to see if the nutraceutical can induce the alternative splicing of SRSF3. At the same time, to see the potential impair on the protein level and on 3D structure.

Materials and Methods

Reverse Transcriptase PCR:
THP1 cell RNA samples were subjected to the reverse transcriptase PCR following QIAGEN One-Step RT-PCR Kit instructions. Temperature cycles as follow: one cycle of 50°C for 30min and 95°C for 15min, 94°C for 30 s, 57°C for 30s, and 72°C for 60s, and a final extension of 72°C for 10 min followed by a final hold at 4°C. Cycle number was 50. The total volume of the PCR system is 10ul.

Electrophoresis:
2ul of loading dye was added to each RT-PCR product. 4ul of each product was running on a 1% agarose gel, and electrophoresis was performed at 160 V. Band intensities were visualized by Ethidium bromide in a UV trans-illuminator (BioRad). 100 bp marker was used to to measure the size of bands.

PCR products purification:
The targeted PCR products were cut and extracted by QIAquick Gel Extraction Kit (QIAGEN) following the manufacturer’s instructions and subsequently sequenced by Sanger sequencing performed by GENEWIZ®.

Sequence analyzing:
Identify the sequencing results by NCBI nucleotide Basic Local Alignment Search Tool (BLAST). Translate mRNA sequence into amino acid by ExPASy translate tool. Align two amino acid sequences by MUSCLE (Multiple Sequence Comparison by Log-Expectation). 3D structure models were provided by I-TASSER server Internet service and then adjusted by Tony.

Results

In order to detect all possible transcript variants of SRSF3, I used the primer pairs that forward from the beginning of exon 2 and reverse from the exon 6 (Fig.1).

PCR was performed on untreated THP1 cell RNA sample and on blessed thistle nutraceutical treated THP1 cell RNA sample. The treated cells show alternative splicing. By Sanger sequencing for every bands and analyzing, I found that band a and b are transcript variant 1. Band c is identical to transcript variant 2 of SRSF3 that has an insertion. And the other band d is the hybrid of those two variants (Fig.2).

In addition, I want to see if the insertion would impair the protein level, I did alignment for isoform 1 and isoform 2. It is shown in figure 3 that they have same RRM domain whereas isoform 2 has shorter RS region than isoform 1with some different amino acids.

The predicted 3D structures are as shown in figure 4. The red colors show the amino acids that are the same. The green and blue colors are the amino acids that are different in each isoform. It is clear that the 3D structures of isoform 2 is different from isoform 1, which may result in different intracellular function.

Discussion

Because of significant roles of SRSF3 in mRNA splicing of other genes, the alternative splicing and isoforms of itself could also be important for other proteins synthesis and thus for maintaining cells in a steady state. In this study, we found that under the treatment of blessed thistle nutraceutical, the splicing pattern has changed that transcript variant 2 increased in treated cells. The transcript variants of SRSF3 has a 456 base pair insertion which could code for another 152 amino acid. However, there exists a stop codon in the insertion that creates an early stop of the translation process, result in a truncated protein. And Because the RS region is shortened, which normally regulates protein to protein interactions and sub-cellular translocation, it may alter the function of the resulting protein.Thus for this project, the further study could focus on the possible expression of isoform2 in cells.

References

[1] Howard JM1, Sanford JR. (2015)The RNAissance family: SR proteins as multifaceted regulators of
gene expression. Wiley Interdiscip Rev RNA.
[2] Huang Y. Steitz J.A. (2001) Splicing factors SRp20 and 9G8 promote the nucleocytoplasmic export
of mRNA. Mol Cell.
[3] Anczukow O, Krainer AR. (2016) Splicing-factor alterations in cancers. RNA.
[4] Jumaa H, Wei G, Nielsen PJ. (1999) Blastocyst formation is blocked in mouse embryos lacking the
splicing factor SRp20. Curr Biol.

Figures


Figure 1-Alternatively spliced transcripts of SRSF3 observed in this study and the locations of primer pairs. The boxes refer to exons. Colored regions (green and blue) can be translated into amino acid sequences. The primers location are as shown in transcript variant 1 and the same in variant 2. Scale length: 500 bases.


Figure 2-SRSF3 alternative splicing in THP1 cell induced by different chemical components. Band a is transcript variant 1. Band b is transcript variant 2. The other bands are hybrids.


Figure 3-Alignment of predicted amino acid sequence of isoform 1 and isoform 2. The amino acids sequences corresponding to RRS motif and RS domain are as shown.


Figure 4-3D structure of isoform 1 and isoform2 of SRSF3.


Abstract

Serine/Arginine-rich splicing factor 3 (SRSF3) is a member of SR protein family that participates in RNA splicing. It is previously known as SRp20 and contains a RNA-binding domain at N-terminal followed by a RS (Arginine and Serine) rich region at the C-terminal. SRSF3 has two transcript variants. The variant 1 has six exon and codes for 164 amino acids. Variant 2 has a 456 base pair insertion. In addition to splicing function, SR proteins are implicated involved in many other roles including RNA transcription, export, translation and decay. This project shows that the nutraceutical derived from Blessed thistle can alter the splicing pattern of SRSF3, increase the production of transcript variant 2 in THP1 cell line, which has different 3D structure from transcript variant 1.

Full Paper

Acknowledgments

I would like to thank Dr. Rubin for his expert guidance for this project. I would also like to thank the two teaching assistants, Anthony Evans and Faaria Fasih-Ahmad, for their infinite patience and assistance. Thanks also go to Dr. Wei for his help.


This document was last modified 05/15/2018.
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