THE ROLE OF EXON B OF THE 5’ UNTRANSLATED REGION OF SURFACTANT PROTEIN A IN THE REGULATION OF EXPRESSION
Open Access
- Author:
- Simmons, Brett Patrick
- Graduate Program:
- Genetics
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- None
- Committee Members:
- Joanna Floros, Thesis Advisor/Co-Advisor
- Keywords:
- Surfactant Protein A
Translation - Abstract:
- Gene regulation is a complex and multifaceted process, and both coding and non-coding sequences are capable of regulatory function. The 5’ untranslated region (UTR) of a gene can have a significant effect on regulation at the levels of transcription and translation. Post-transcription, the 5’ UTR is capable of conferring mRNA stability, providing binding sites for translation machinery, containing degradation and/or localization signals, and other functions dependent on tertiary RNA structure and RNA-protein structure. One gene in which the 5’ UTR is essential for proper control of protein expression is Surfactant Protein A (SP-A). SP-A is one of four pulmonary surfactant-associated proteins, all of which are encoded by separate genes. A thin liquid layer covers the distal airspaces or alveolar spaces. Pulmonary surfactant is found between the air-liquid interface of the distal airspaces and its function is to reduce surface tension at low lung volumes and prevent alveolar collapse. Pulmonary surfactant is comprised of proteins and lipids that together play a vital role in lung function and host defense. Furthermore, aberrant expression of SP-A is a characteristic of many pulmonary diseases and disorders, including asthma, respiratory distress syndrome, and cancer, among others. Previous studies have indicated that exon B of the SP-A 5’ UTR may be a potent cis-acting element of post-transcriptional and translational regulation. Using the luciferase assay and quantitative real-time PCR, the translational enhancer capacity of this 5’ UTR exon was assessed to investigate the hypothesis that the 30-basepair exon B sequence is a cis-acting translational enhancer. Transcription was normalized by using an SV40 promoter and through the calculation of a translational index (activity divided by relative mRNA content). To investigate the function of exon B in the context of the whole SP-A 5’ UTR, exon B was deleted from the 5’ UTR. In another construct, the exon B sequence was replaced with a 30-basepair random sequence. Compared with the whole SP-A 5’ UTR construct, these two constructs, which lack the exon B sequence, showed reduced luciferase activity. This indicated that exon B is capable of increasing translation activity and could have resulted from an increase in stability, altered localization patterns, or an increase in translation efficiency. To determine whether exon B acts as a translational enhancer in the context of other 5’ UTR sequences, constructs that each contained one of two independent guest 5’ UTRs cloned downstream of the B exon were used in a guest 5’ UTR study. The SP-B and SP-D 5’ UTRs functioned as guest 5’ UTRs. While the size of the leader sequence appeared to have little effect on translation, constructs containing the complete exon B sequence were found to have activities twice as high as the activities of 5’ UTR constructs containing only the guest 5’ UTRs. Constructs containing exon B also exhibited twice as much activity as those containing random 30-nucleotide sequences in place of exon B. This indicated that exon B is a translational enhancer and functions in a sequence-specific manner. Furthermore, the combination of the expression and real time PCR data suggest that SP-A 5’ UTR exon B enhances expression by improving translational efficiency. A finding unrelated to SP-A 5’ UTR exon B was that the SP-D 5’ UTR is stronger than the SP-B 5’ UTR, meaning that it enhanced expression by a larger amount when the luciferase assay was used to measure activity. Using a series of deletion mutations, as well as other site-directed mutagenesis changes, the region of this noncoding exon that is responsible for the expression enhancement was investigated. For this, 5 basepairs were deleted at a time within the B exon of a complete SP-A 5’ UTR-luciferase construct, and these deletion mutation constructs were transfected into NCI-H441 cells. Luciferase assay results indicated that a 15-nucleotide region appeared to be important for exon B’s translational enhancement. Translation is a complex and dynamic biological process and is regulated at several levels and by various means. While elucidating the precise mechanisms of SP-A translational control is beyond the scope of the present study, the resources and technology required to advance our understanding of these processes are available. Future studies may reveal the trans-acting factors necessary for SP-A translation and how the process of regulating SP-A expression works from nuclear mRNA export to translation termination.