The detection of tissue specific alternative splicing of mouse CSPP




yiming yu

Introduction

mCSPP may play an important role in cell cycle progression and cell division. The identification of CSPP in mice and the study of its function was first published in 2009. Scientists successfully cloned the cDNA for two alternatively spliced forms of mCSPP: mCSPP-1, and mCSPP-2. They determined their cellular localization with GFP-tagged mCSPP-1, and -2 (see Fig.1). The image shows that GFP-mCSPP-1 localized to the spindle pole and central spindle. GFP-mCSPP-2 also localized to the central spindle in anaphase. However, GFP-CSPP-2 predominantly localized to the spindle microtubules in metaphase. Moreover, it was reported that mCSPP interact with MyoGEF, a guanine nucleotide exchange factor that localizes to the central spindle and cleavage furrow. Depletion of mCSPP or MyoGEF by RNA-interference will causes defects in mitosis and cytokinesis, such as metaphase arrest and furrow regression. We know that mCSPP is alternatively spliced and some alternatively spliced variants have different functions in cell cycle progression and cell division.
In this study, we wanted to see if we could detect alternative splicing of mCSPP. We also wanted to see if mCSPP is differentially spliced in different tissues.

Materials and Methods

Lung, kidney and heart tissues were isolated from a mouse. Tissues were homogenized and RNA was extracted using the RNeasy® Plus Mini Kit (QIAGEN). Reverse-Transcriptase-PCR (RT-PCR) was used to amplify different regions of the mCSPP transcripts. RT-PCR products were visualized by gel electrophoresis on a 1% agarose gel. Gel extraction was performed using the QIAquick® Gel Extraction Kit. Purified PCR products were sent for sequence analysis to GENEWIZ®. Sequences were aligned and analyzed using NCBI BLAST.

Results

There is known alternative splicing pattern of exon 18 which contains 153 nucleotide base pairs. mCSPP is not expected to splice between exons 6 and exon 12, so I use this region as an internal control (see Fig.2).
Five pairs of primers were designed: F16R19, F16R19’, F10R12, F6R8, F6R8’. The position of each forward and reverse pirmers as well as size of products are shown (see Fig.3). F16R19 and F16’R19’ were used to amplify the sequence between exon 16 and exon 19 while F10R12, F6R8, and F6R8’ were used to amplify conserved sequence from exon 10 to exon 12 and from exon 6 to exon 8.
The RT-PCR was performed with the RNA collected from mouse kidney, lung and heart. In kidney and lung four bands were observed within primers from exon 16 to exon 19. They were purified and sent out for sequencing. Two bands with larger size were confirmed that they are the alignment of exon 16, exon 17, exon 18 and exon 19 (see Fig.4, lane 1 and 2).
On the contrary, two bands with smaller size were confirmed that they are the alignment of exon 16, exon 17 and exon 19 (see Fig.4 lane 1and 2). One band was obverted within primers from exon10 to exon 12. The sequence analysis shows that it contains exon 10, exon 11 and exon 12 (Fig.4 lane 3). Totally two bands were obverted within primers from exon 6 to exon 8. And sequencing result shows that they are the alignment of exon 6, exon 7 and exon 8 (Fig.4 lane 4 and 5). However, in heart, only two larger bands were visualized within primers from exon 16 to exon 19 (Fig.4 lane 13 and 14 ).
Together, these results show that we detected the alternative splicing pattern happens in exon 18 in mouse kidney and lung but not in heart.

Discussion

These results indicate that we detected the alternative splicing pattern of the exon 18 of mCSPP in mouse kidney and lung but not in heart. One possibility is that there is no expression of transcription variant which does not containing exon 18, another possibility is that there are expression of transcription variant containing exon 18 in heart but in low level, because it is clear that the expressional level of transcription variants with exon 18 is higher than the transcription variants without exon 18. So, in order to test which explanation is reasonable, we need to further test the alternative splicing pattern of mCSPP in heart.
Together with some previous studies, we can only hypothesize that the spliced out of exon 18 will affect the localization of mCSPP during cell division because no study so far focused on the biological meaning of these spliced out regions.
This data shows that mCSPP is differentially spliced in different tissues.

Figures


Figure 1-The localization of mCSPP-1.


Figure 2-The structure and alternative splicing pattern of mCSPP gene.


Figure 3-The position of each primer pairs and the size of products.


Figure 4-RT-PCR and sequencing results with RNA from mouse kidney (lane 1-6), lung (lane 7-12), and heart (lane13-18). Six primer pairs(including mGAPDH) were used.


Centrosome/Spindle Pole-associated Protein (CSPP) was first identified by Dr. Sebastian Patzke and Dr. Trond Stokke in 2006. It is a protein that is concentrated at the spindle pole and central spindle during mitosis and cytokinesis and plays an important role in cell cycle progression. In 2009, scientists discovered the expression of CSPP in mouse and identified several different isoforms of mouse CSPP (mCSPP) such as mCSPP-1 and mCSPP-2. These isoforms are the result of alternative splicing of mCSPP. As of now, there is no report of the detection of tissue specific alternative splicing of mouse CSPP. In this study, we detected the alternative splicing of exon 18 in the mouse CSPP using RT-PCR. In this study, we found transcripts of mCSPP that both contained and lacked exon 18 in the kidney and lung tissue but only transcripts that contain exon 18 in heart tissue.

Full Paper

Acknowledgments

I would like to thank Dr. Wei for providing many kinds of mouse tissues and mRNA samples as well as useful suggestions. I would like to thank Anthony Evans and Farria Fasih-Ahmad for their fantastic work as Teaching Assistants and for their never-ending patience and efforts they put in to make this project undergo successfully. Finally, I would like to thank Dr. Rubin for his guidance and for making this project possible.


This document was last modified 05/15/2018.
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