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).
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.
-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.
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.
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