Identification of cognate RNA for the RNA-binding protein, RbpA, from Synechococcus sp. PCC 7942

Strozen, Timothy G. (2005) Identification of cognate RNA for the RNA-binding protein, RbpA, from Synechococcus sp. PCC 7942. Masters thesis, Memorial University of Newfoundland.

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Abstract

The role RRM-type RNA-binding proteins (Rbps) perform in the cyanobacterial cell is unknown. However, evidence suggests that RRM-type RNA-binding proteins that contain a glycine-rich C-terminal auxiliary domain such as RNA-binding protein A from Synechococcus sp. PCC 7942 are involved in the cyanobacterial cold-shock response. In this investigation, the genomic SELEX technique was used to gain insight into the function of cold-inducible RbpA in the cyanobacterial cell by determining the nucleic acid binding specificity of the protein and by identifying the genes potentially regulated by RbpA. -- The genomic SELEX technique involved use of a dsDNA library that contained 38-88 bp portions of Synechococcus 7942 genomic DNA sequence flanked by fixed DNA sequences. Representation of the entire Synechococcus 7942 genome in the genomic SELEX library was verified by nested-PCR analysis of a 43 base portion of the rbpB (RNA-binding protein B) gene. The library contained, in a staggered arrangement, molecules whose genomic portion terminated at 8 of the possible 13 nucleotides that correspond to bases 322 to 334 of the rbpB gene. These results indicated that if the genome was as equally represented in the library as that of the 13 base region of rbpB, then the genomic SELEX library would contain 3.38 x 10⁶ different molecules. This number of library molecules corresponds to one molecule per 1.6 bases in the Synechococcus 7942 genome and thereby provided evidence that the library was a sufficient representation of the Synechococcus 7942 genome and could be used in the genomic SELEX procedure. -- The genomic SELEX procedure involved multiple rounds of the same basic steps. The library was transcribed into RNA, RNA was mixed with N-terminal histidine-tagged RbpA (H6RbpA) in a protein-RNA binding reaction. Isolation of H6RbpA-RNA complexes was accomplished by Nt2-NTA metal chelate chromatography and the RNA molecules retained by this purification method were reverse-transcribed and PCR amplified to generate the dsDNA library used in the next round of selection. -- To identify the nucleic acid binding specificity of RbpA, representative clones from the genomic SELEX library were sequenced after rounds 10 and 14 of selection. Following round 10 of selection, the amplified dsDNA was cloned into pUC19 and sequenced. Due to a lack of obvious sequence homology of the molecules selected after round 10 of SELEX, an additional 4 rounds of selection were performed under conditions of increased stringency. These conditions included an increase in salt concentration from 75mM to 150mM and a decrease in the RNA/protein ratio from 200:1 to 1:1. These conditions greatly enhanced selection, as evidenced by a substantial increase in nucleic acid sequence homology of representative members of the round 14 SELEX library. RNA selected in both rounds were of three types, RNA molecules poor in G/U residues (less than 50% G/U), rich in G/U (greater than 50% G/U) and G/U very rich sequences (greater than 90% G/U). Existence of G/U poor sequences was suprising because like many RRM-type RNA-binding proteins, RbpA has been shown to have a strong binding preference for GTP and UTP RNA homopolymers. Sequence homology analysis of the most homologous group of RNA molecules, the round 14 G/U rich RNA, identified a highly conserved putative RbpA consensus binding sequence 5'UGAAUGGGAGGUG 3'. The six 3' terminal ribonucleotides could be the most important nucleotides of the putative consensus sequence due to the similarity of the 5' GAGGUG 3' sequence with a sequence 5' GUGGUG 3' present in many GIU very rich RNA sequences. -- Comparative genomic sequence analysis of all sequences retained in rounds I 0 and 14 of genomic SELEX allowed me to determine the genes whose RNA products are potentially regulated by RbpA via a binding interaction. Comparative BLAST analysis of the SELEX DNA sequences with that of the Synechococcus 7942 genomic sequence identified many genes. These include cold-shock inducible genes such dsg, putP and sodB that encode fatty acid desaturase, proline permease and superoxide dismutase respectively. Other interesting genes identified by comparative analysis include two transcription regulator proteins that contain conserved helix-tum-helix motifs and ntrB that encodes a nitrate reductase. Interestingly, some sequences identified by genomic SELEX corresponded to a sequence on the non-coding strand within the open reading frame of a gene. This result suggests that if RbpA is involved in regulating expression of these genes, it would involve the expression of a cis-encoded RNA molecule. This mechanism of gene regulation has not been studied extensively in prokaryotes and has not yet been characterized in cyanobacteria, however it has been identified in the eukaryotes. These results suggest that RbpA is involved in regulation of many genes that in some cases could involve a previously uncharacterized mechanism of gene regulation in cyanobacteria.

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