The role of splicing factors and small nuclear RNAs in spliceosomal formation

Protein coding genes are comprised of protein-coding exons and non-protein-coding introns. The process of splicing involves removal of the introns and joining of the exons to form a mature messenger RNA, which subsequently undergoes translation into polypeptide. The spliceosome is a large, RNA/protein assembly of five small nuclear RNAs as well as over 300 proteins, which catalyzes intron removal and exon ligation. The selection of specific exons for inclusion in the mature messenger RNA is spatiotemporally regulated and results in production of an enormous diversity of polypeptides from a single gene locus. This phenomenon, known as alternative splicing, is regulated, in part, by protein splicing factors, which target the spliceosome to exon/intron boundaries. The first part of my dissertation (Chapters II and III) focuses on the discovery and characterization of the 45 kilodalton FK506 binding protein (FKBP45), which I discovered in the silk moth, Bombyx mori, as a U1 small nuclear RNA binding protein. This protein family binds the immunosuppressants FK506 and rapamycin and contains peptidyl-prolyl cis-trans isomerase activity, which converts polypeptides from cis to trans about a proline residue. This is the first time that an FKBP has been identified in the spliceosome. The second section of my dissertation (Chapters IV, V, VI and VII) is an investigation of the potential role of small nuclear RNA sequence variants in the control of splicing. I identified 46 copies of small nuclear RNAs in the 6X whole genome shotgun of the Bombyx mori p50T strain. These variants may play a role in differential binding of specific proteins that mediate alternative splicing. Along these lines, further investigation of U2 snRNA sequence variants in Bombyx mori demonstrated that some U2 snRNAs preferentially assemble into high molecular weight spliceosomal complexes over others. Expression of snRNA variants may represent another mechanism by which the cell is able to fine tune the splicing process.

Absolute configuration and biosynthesis of pahayokolide A from Lyngbya sp. Strain 15-2 of the Florida Everglades

Pahayokolides A-D are cytotoxic cyclic polypeptides produced by the freshwater cyanobacterium Lyngbya sp. strain 15-2 that possess an unusual β-amino acid, 3-amino-2,5,7,8-tetrahydroxy-10-methylundecanoic acid (Athmu). The absolute configuration of pahayokolides A-D was determined using advanced Marfey’s method. It was also confirmed that a pendant N-acetyl- N-methyl leucine moiety in pahayokolide A was absent in pahayokolides B and pahayokolides C-D were conformers of pahayokolide A. Feeding experiments indicated that the biosynthesis of the Athmu sidechain arises from leucine or α-ketoisovalerate, however could not be further extended by three rounds of condensation with malonate units. Putative four peptide and one unique polyketide synthetases in Lyngbya sp. strain 15-2 were identified by using a PCR method and degenerate primers derived from conserved core sequences of known NRPSs and PKSs. Identification of one unique KS domain conflicted with the logic rule that the long side chain of Athmu was assembled by three rounds of ketide extensions if PKSs were involved. A gene cluster (pah) encoding a peptide synthetase putatively producing pahayokolide was cloned, partially sequenced and characterized. Seven modules of the non-ribosomal peptide synthetase (NRPS) were identified. Ten additional opening reading frames (ORFs) were found, responsible for peptide resistance, transport and degradation. Although the predicted substrate specificities of NRPS agreed with the structure of pahayokolide A partially, the disagreement could be explained. However, no PKS gene was found in the pah gene cluster.

The Role of Splicing Factors and Small Nuclear RNAS in Spliceosomal Formation

Protein coding genes are comprised of protein-coding exons and non-protein-coding introns. The process of splicing involves removal of the introns and joining of the exons to form a mature messenger RNA, which subsequently undergoes translation into polypeptide. The spliceosome is a large, RNA/protein assembly of five small nuclear RNAs as well as over 300 proteins, which catalyzes intron removal and exon ligation. The selection of specific exons for inclusion in the mature messenger RNA is spatio-temporally regulated and results in production of an enormous diversity of polypeptides from a single gene locus. This phenomenon, known as alternative splicing, is regulated, in part, by protein splicing factors, which target the spliceosome to exon/intron boundaries. The first part of my dissertation (Chapters II and III) focuses on the discovery and characterization of the 45 kilodalton FK506 binding protein (FKBP45), which I discovered in the silk moth, Bombyx mori, as a U1 small nuclear RNA binding protein. This protein family binds the immunosuppressants FK506 and rapamycin and contains peptidyl-prolyl cis-trans isomerase activity, which converts polypeptides from cis to trans about a proline residue. This is the first time that an FKBP has been identified in the spliceosome. The second section of my dissertation (Chapters IV, V, VI and VII) is an investigation of the potential role of small nuclear RNA sequence variants in the control of splicing. I identified 46 copies of small nuclear RNAs in the 6X whole genome shotgun of the Bombyx mori p50T strain. These variants may play a role in differential binding of specific proteins that mediate alternative splicing. Along these lines, further investigation of U2 snRNA sequence variants in Bombyx mori demonstrated that some U2 snRNAs preferentially assemble into high molecular weight spliceosomal complexes over others. Expression of snRNA variants may represent another mechanism by which the cell is able to fine tune the splicing process.

Absolute Configuration and Biosynthesis of Pahayokolide A from Lyngbya sp. Strain 15-2 of the Florida Everglades

Pahayokolides A-D are cytotoxic cyclic polypeptides produced by the freshwater cyanobacterium Lyngbya sp. strain 15-2 that possess an unusual β-amino acid, 3-amino-2,5,7,8-tetrahydroxy-10-methylundecanoic acid (Athmu). The absolute configuration of pahayokolides A-D was determined using advanced Marfey’s method. It was also confirmed that a pendant N-acetyl-N-methyl leucine moiety in pahayokolide A was absent in pahayokolides B and pahayokolides C-D were conformers of pahayokolide A. Feeding experiments indicated that the biosynthesis of the Athmu sidechain arises from leucine or α-ketoisovalerate, however could not be further extended by three rounds of condensation with malonate units. Putative four peptide and one unique polyketide synthetases in Lyngbya sp. strain 15-2 were identified by using a PCR method and degenerate primers derived from conserved core sequences of known NRPSs and PKSs. Identification of one unique KS domain conflicted with the logic rule that the long side chain of Athmu was assembled by three rounds of ketide extensions if PKSs were involved. A gene cluster (pah) encoding a peptide synthetase putatively producing pahayokolide was cloned, partially sequenced and characterized. Seven modules of the non-ribosomal peptide synthetase (NRPS) were identified. Ten additional opening reading frames (ORFs) were found, responsible for peptide resistance, transport and degradation. Although the predicted substrate specificities of NRPS agreed with the structure of pahayokolide A partially, the disagreement could be explained. However, no PKS gene was found in the pah gene cluster.