Caenorhabditis elegans rpn6 2 Alternative m RNA Splicing
Caenorhabditis elegans rpn-6. 2 Alternative m. RNA Splicing Robert L. Owen, Sara A. Moore, Dr. Rebecca L. Seipelt-Thiemann, Biology Department, Middle Tennessee State University, Murfreesboro, TN Introduction Ø Protein quality control is an aggressively studied and researched area of biological science. Ø The rpn-6. 2 gene is an understudied regulatory particle that is required for the assembly of the 26 S subunit proteasome, which is a protein complex that destructs cell pieces that will be broken down and reused for other purposes in the cell. Ø Caenorhabditis elegans is an ideal organism for experimental study because of its transparency, fixed cell count and consistent cell development. Intron 2 36 Ex ed : Tr e at l: ro nt Co on s 36 on Ex ’G (O r de La d Tr e s en e 24 on s at ed : Ex s on Ex Co nt ro l: Ex ed : at Tr e 24 -4 on s 1 14 on Ex l: ro nt 1500 1200 Base 1000 pairs (bps) 500 (bps) 400 300 500 Ideal annealing temperature (58. 5 C) Figure 4 b: Experimental Results: From Promoter #1, alternatively spliced RNA sequences extracted from wild type after treatment with 500 m. M Na. Cl salt stress for 60 minutes (SMART 2017) Figure 2 b: Control: Exons 1 -4, bands at 1600, 1000 Treated: Exons 1 -4, bands at 1600, 1400, 1000 Control: Exons 2 -4, bands at 575, 500 Treated: Exons 2 -4, no bands Intron 4 Intron 3 Co Figure 4 a: Experimental Results: From Promoter #1, alternatively spliced RNA sequences extracted from wild type control (SMART 2017) Figure 2 a: Determined Annealing Temperature Intron 5 s en e ’G (O La d de r de The purpose of this project is to see if alternative splicing occurs for the gene rpn -6. 2. Caenorhabditis elegans wild-type worms were environmentally stressed and compared with control. RNA was then extracted and assayed via electrophoresis. Alternative splicing by inclusion of certain introns were observed. Completely unspliced strands were also observed. It can be considered that under certain environmental stress, rpn-6. 2 is deactivated due to intron inclusion and thus introducing early stop codons. Perhaps this is to allow for another regulatory particle to instead have room to function. In the future, RNA regulatory mechanism assays should be used to further understand the implications of certain introns being including. Ru le r) 60 r (O. 6 ’ ºC Ge ne 58 Ru. 5 le ºC r) 55. 4 ºC 51. 5 ºC 48. 2 º 46 C. 2 ºC Abstract Ru le r) Results Figure 2 c: Control: Exons 3 -6, band at 300 Treated: Exons 1 -4, bands at 300, 450 Intron 1 Figure 4 c: Experimental Results: From Promoter #2, alternatively spliced RNA sequences extracted from wild type control (SMART 2017) Exon 6 Exon 5 Exon 4 Exon 3 Exon 2 Exon 1 Figure 1 a: rpn-6. 2 Gene sequence with both isoforms, a and b (Wormbase 2017) Figure 4 d: Experimental Results: From Promoter #2, alternatively spliced RNA sequences extracted from wild type after treatment with 500 m. M Na. Cl salt stress for 60 minutes (SMART 2017) Key: Figure 1 b: Generic gene structure showing both promoters Conclusions Unknown region Low complexity region PCI/PINT associated module (plays a role in protein binding) Motif in proteasome subunits, Int-6, Nip-1 and TRIP-15 (unknown function) Goal The goal of our experiment is to analyze whether or not alternative splicing occurs in the rpn-6. 2 gene in wild-type C. elegans under normal and environmentally stressed conditions. Coiled Coil Figure 3 a: Theoretical Alternative Splicing Scenarios (SMART 2017) Methods Ø Ø Ø Ø Design exon-specific rpn-6. 2 primers for and understand C. elegans genome using genomic databases Grow bacteria and nematodes Treat nematodes with environmental stressor (salt stress) of 500 m. M salt stress for 50 minutes Isolate RNA Perform Reverse Transcription Polymerase Chain Reaction (RT-PCR) Agarose gel electrophoresis Analyze gene expression (alternative splicing) Forward Reverse Trial #1 Exon 4 Trial #2 Exon 4 Trial #3 Exon 6 Figure 3 b: Primer variations used to compile splicing results across three trials Ø The result of the RT-PCR indicates that alternative splicing (including certain introns) and unsplicing did occur in all tested regions under stressed and normal conditions. Ø Control groups: promoter 1 included ref. seq. and all unspliced region, promoter 2 included intron 2 and ref. seq. Ø Treated groups: promoter 1 showed completely unspliced, ref. seq. and included intron 1; promoter 2 showed exon 3 - exon 6 unspliced and ref. seq. Future Directions Ø Use RNA regulatory mechanism assays for Intron 2 to see if there is a difference in including intron 2 versus splicing it out, and to determine why ribosome ignores the three start codons within Exon 2 when using Promoter 2. Ø Learn why Isoform b does not start translation until Exon 3. Ø Determine what conditions cause Promoter 2 to be used. References • Ciccarelli, Francesca; Izaurralde, Elisa; Bork, Peer. The PAM domain, a multi-protein complex-associated module with an all-alpha-helix fold. BMC Informatics [Internet]. 2003 [cited 2017 Dec. 5]; 10. 1186/1471 -2105 -4 -64. Available from: https: //doi. org/10. 1186/1471 -2105 -4 -64 • Ex. PASy Bioinformatics Resource Portal Translate Tool [Internet]. Swiss Institute of Bioinformatics 2017 [cited 2017 December 5]. Available from: https: //web. expasy. org/translate • Glickman MH, Rubin DM, Fried VA, Finley D. The regulatory particle of the Saccharomyces cerevisiae proteasome. Mol Cell Biology [Internet]. 1998 [cited 2017 Dec. 5]; 18(6): 3149– 3162. Available from: https: //www. ncbi. nlm. nih. gov/pmc/articles/PMC 108897/ • O'Gene. Ruler DNA Ladder Mix, ready-to-use [Internet]. Thermo Scientific 2016 Nov. 30 [cited 2017 Nov. 16]. Available from: https: //assets. thermofisher. com/TFS-Assets/LSG/manuals/MAN 0013034_OGene. Ruler_DNALadder_RTU_UG. pdf • Sampuda, Katherine M. , Mason Riley, Lynn Boyd. Stress induced nuclear granules form in response to accumulation of misfolded proteins in Caenorhabditis elegans. BMC Cell Biology [Internet]. 2017 [cited 2017 Oct. 4]; 18: 18. Available from: https: //dx. doi. org/10. 1186%2 Fs 12860 -017 -0136 -x • SMART [Internet]. Simple Modular Architecture Research Tool 2017 [cited 2017 December 5]. Available from: http: //smart. emblheidelberg. de • wormbase. org [Internet]. Wormbase 2017, Worm. Base ID: WBGene 00010309 [cited 2017 December 5]. Available from: http: //www. wormbase. org/species/c_elegans/gene/WBGene 00010309#0 -9 g-3
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