Expression Patterns of Splicing Factors in Four Cell Lines

Introduction

In eukaryotes, the presence of introns within the transcribed region of a gene adds a level of complexity to the expression patterning of genes. Correct splicing of introns out of an immature RNA is required to ensure that a proper message will exit the nucleus and be translated into a functional protein. Heterogeneous nuclear ribonucleoproteins (hnrp) are the protein molecules that are involved in this function. Complexed with RNA, these proteins have also been implicated in the shuttling of mature messenger RNAs to the cytoplasm and may also play roles in their subsequent translation (Caporali et al. 2005).

Hnrp molecules are the most abundant proteins found in the nucleus; interestingly, most members of this family possess additional functions besides the ability to splice pre-mRNA. The hnrpC gene encodes two known isoforms, C1 and C2, which form a tetramer containing three units of the C1 isoform and one unit of the C2 isoform (Nakielny and Dreyfuss 1996). HnrpC is required for the assembly of the 40S hnRNP particle (Huang et al. 1994). A second member of this family, hnrpM comprises the N-acetylglucosamine-specific receptor that has been implicated in the recycling of the glycoprotein thyroid hormone precursor thyroglobulin. Importantly, elevated blood levels of thyroglobulin have been associated with thyroid cancer (Bajenova et al. 2003). With regards to a third member of this family, hnrpR, a previous study conducted by J Huang et al. (2005) established that expression of two isoforms of hnrpR vary widely and may be involved in neural development. Specifically, whereas the R1 isoform is expressed robustly in nearly all tissues, R2 is much more lowly expressed and is found only in neural tissues (Hassfield et al. 1998). Finally, hnrpU also possesses a scaffold-associated region (SAR)-specific DNA-binding domain in addition to the RNA binding domain characteristic of all hnrp proteins (Gupta et al. 1998). HnrpU plays a significant role in the apoptotic pathway as it is cleaved in the caspase pathway and subsequently can no longer bind DNA (Kipp et al. 2000). Thus, these proteins exhibit a wide variety of functions that make each a valuable marker in the assessment of the health of a cell.

HEK293 is a cell line derived from embryonic kidney cells that were transformed using adenovirus DNA. Similarly, CF2789 cells are derived from lymphoblast cells transformed via infection with the Epstein Barr virus. Using these cell lines as well as the nonmalignant LAI-5S neuroblastoma cell line and the malignant LAI-55N neuroblastoma cell line, the aim of the study presented here was to assess the expression patterns of these four splicing factors to more fully grasp the gene expression pattern of these cells.

Figures

Figure 1-Analysis of RT-PCR products. RT-PCR was performed as described, and the resultant products were run on a 1.5% agarose gel. The labels on the left indicate the mRNA species that was reverse transcribed and amplified. The labels on the top indicate the cell line from which the RNA was extracted and subjected to RT-PCR. Negative control indicates the absence of added RNA in the RT-PCR reaction.

Figure 2-Alignment of sequenced RT-PCR products with published sequences. RT-PCR products were purified and prepared for sequencing as described. The resulting sequences were then compared to established sequences using BLAST. The RT-PCR product obtained using the hnrpC primer set (red) matched the established hnrpC mRNA sequence (accession number NM_031314). The hnrpM product (blue) matched the published hnrpM mRNA sequence (NM_005968); the hnrpR product (purple) matched the published hnrpR mRNA sequence (NM_005826), and the hnrpU product (green) matched the established hnrpU sequence (NM_031844). The GAPDH product (black) completely matched the known GAPDH mRNA sequence (NM_002046.3). Parenthetical terms shown in the alignments indicate the primers used to obtain the respective RT-PCR product sequence (i.e. F indicates the forward primer, and R indicates the reverse primer).

-In this study, the expression patterns of four hnrp proteins were assessed in four cell lines, and the following phenomena were observed:

-HnrpC is weakly expressed in the LAI-55N cell line and is comparably expressed in the HEK293, LAI-5S, and CF2789 cell lines.

-HnrpU is weakly expressed in the LAI-5S cell line, intermediately expressed in the LAI-55N cell line, and most highly expressed in the HEK293 and CF2789 cell lines.

-HnrpM and HnrpU are comparably expressed in all four cell lines.

-Sequencing data indicates that the primers used in this experiment amplified the intended sequences.

Future Prospects:
-Each of the four hnrps analyzed here have been shown in previous studies to be involved in various metabolic pathways. What role does this play in determining the expression pattern of these splicing factors in the cell lines studied here as well as in other cell types?

Please refer to the attached full paper for more details.

Full Paper

Acknowledgments

I would like to thank Dr. Sylvia Anderson who kindly provided materials that allowed me to have a successful project. I am also grateful to teaching assistants Leleesha and Joe who offered countless pieces of advice. Finally, I would like to thank Dr. Berish Rubin for allowing this project to take place.

This document was last modified 05/09/2007.
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