TC001087
Fly Ortholog:
LRRK
Student Name:
Rebekah Mohn and Jessica Schmeling
School Name:
Miami University
Year of Study:
2017
Gene Information:
Protein features (e.g. Zn finger, transmembrane, signal transducer, transcription factor, etc):
LRRK2 gene codes for a large Ser/Thr kinase (2527 amino acids) with two conserved domains: Roc (Ras in complex proteins) domain belonging to the Ras/GTPase superfamily and a COR domain (C-terminal of Roc). Three additional conserved domains have been described: leucine rich repeat (LRR); a tyrosine kinase catalytic domain (MAPKKK); WD40 domain.
Biological Function:
Leucine-rich repeat kinase (Lrrk) encodes a large Ser/Thr kinase. Lrrk roles include mRNA translational control, cytoskeleton regulation, vesicle trafficking, autophagy, and immune response.
In humans, roles of LRRK2 include neurogenesis and neurite outgrowth, cytoskeleton assembly, endocytosis/vesicle trafficking, and autophagy coordination. The WD40 domain is crucial in several basic cell functions such as vesicle sorting during endocytosis and exocytosis of synaptic vesicles, vesicle-mediated transport, and cytoskeleton assembly.
iBeetle Link:
Tribolium Transcript: 5' UTR:
acaattaaactaacctctaatccagcagtcaaaataaggacatttttgtatttctcaatcgttgcgaaattattatcgttgaaacgcttcgcggtttttgcacgaaattgtgttagtttccgcgtttgtgtaatgtaggtgttgggagcgttatcgatcgccaca
Tribolium Transcript: CDS:
atgttggattcttcgcctgaagacgaagattttccaggccggctcctacaccaggccgcgttatgggacaacacacatttactcatggacttgctagaagggggccaacaggccttcctgaactcgcaggacagttgggggagaacccccctacatgcagctgctattaatgataattcagggtgtttgacaattttactaaatgcaggggccgatcctaacattcggtgcggcccaagaggcgaccagaagattcctttgcatttaagtgccgaacatgggcataacagtaatgtctcaattttactgcagcatgattcagatattcaaattctggatgcgaatggaatgagtgctctcgatctggctgaaaaagcggggcatgtgaagtgtgtaacactattgaaagaagctgtggatgcgagagaaaaagcgagacttgatattcatgcagcgcttagggacgcctgctgccagggagatgtgaccactgtttcttcacttttggcctctttgtccaaagatgccgaacttattgttaacatggctccgagcggagcgagtactttactatttacagcttgccagacggggtgcaaagatatcgtcaaggtactgctagaccacggggccgatggccggtaccaccctgtcaccaaatattcgccgctttacattgcctgccactatggacatagagatattgtagagatgctgcttttgcgatttcccgaactcatacagcaacacactgtggaacgatggctcccgatccacgccagcgccatcaatggtcacgtggacgtgttggaactacttttccactttccgtacccttcgcacgttttgcgcaaatttagagatggcagcgaacagtgggaatattttatgccttttgacatcaatgagcgagacgccactggccaaaatattttatacatggcttgtttagtaggaaataagaaacttttagatgtgatattaaaatttaaagttaaagctactagaatccaagcggacgatgaggtaacgcctgtttctgaaagtagtgtggttttgagtcctaccaagcgtcgcatttcggatggaattcagtcaattttgtccaaattaaatctatcgagggataattcaagcgaaagcgatgttgacccgaatagttctatgatttgcccgttacggattgatatgtattgtaataataacacggagacggccctccatgctgctattaaaggcaagcactacgatatagcactggctttgttgaatgctggggcgaattcaaacgctgtgattaaagcctatcacgacttggaaaatttgagttgttgttccgttgaggaagactcaaactacggtcaatcgaccggtttagtcgaagcttgtcgcaaccgtgacgttcctatagtcgatcttttactcaaatacggcgcccgagacgacgaatgtaaagccctatcggtggctgtacaaaacaaagacgagacactaacagccaaactcttatcgacaaaagcccacccggaccctgagttcaaaatcaacaaaaaagcaatgacggaaaacgtgaactcgtcccaatttacaattttctcaaacgttaccaacttgacctacagcgccctttttcccaacacaccgaccatgatcaactggcacaaccaacggtgcaacctcacccaaatacgcacccagtggttgaccgacgcagctttgcacctcaaccagaaactcaaacaaaatccccgaagctacgacatcgccttatacgcaattacacgtctcgatgtctctaacaacaacctaacgtccataccactggctgtgtttcaattgtatagtctgaggtacttgaatattgggcaaaacaaaatcgagaaattaccaactccggttgcctcgcccaccaagaaaggtgtgcgcaaaaaaaacgctaacgaagaattgaattattcgtgccccgtcttggaggagttatacttgcaagacaaccgacttgatcacatcccggacgctatatttcggttaccatcgttggtaactctagtcgtgtccaataataagttgcagcaattgtcgtataatatgtggctagccccgaaattgaaggaattgaacgcttcgttcaacttgcttaaagaactaccaactaacatgtccgagacgcaagaggagtgcgataaaatgagcgtaagctcgagcgatagccagttgtccagtcattgtgaccacgactcggagagcgaggtccgatgcgactttgacttggactccaagtatctctcaagcgacgaaaacagccaatgcgtcaagtacaggaaaaaagagcgctcttttattcaatttgacctggctaagcaccacatctggagcaagaacgtcgaagtgaccgagcaaattctccacactgacgagaccgtgactgagcagtcgtccaaactgtcggccctcaacttggcccacaacctttttacatcaattccagtggtgttaccgtgcctggcggtcaatttgactcgtttgaacatggcttttaactcgctgaggtcgatgtcgcacatcaccagctaccccagttctttgaaacagctggatttgagtcacaatcagatcacttgttggccaagtttgccgcaagtcgaggcgcaggatgtgatggagttggcgaacattgcgtgctacgcgagcagtgtggggtcgagggagaaggtttcggggattgctgcccgtagacagccgggccggtcggtccggaacacggttctgcattcggtgtgttcgcaccgaagacacttgaagttggatagcttgcggacgctgattttggccgataatcagttgagccggattcagttaagtacggatgatgatggggatgttagtgctgtcgaggaggatgatctcgaacgtcgcaaagaaattaatacgcattttgaagatgactttatttgtgtatctttttccaaggttgtggggaatccggcggcggggaaggcacggttgatgttccccaatttatcaatgcttgatgttagtaacaataatttgaaagaaattcctataaatatcaacgaactcagtaacttgtccgtcctaaatataagtgggaatctagacgttaatgaactcccaccacaaatgggtctactatcgcgcttatggaatctcaacacgcgcggttgttctttacaagatccgttaaaatcgatgattgatagtaaaaagtacaaaacaatggatattgtgggttatttgaaatcggtgcttgaggatgcgagaccttacgctcgcatgaaattaatgatagtgggagtgcaaggaattgggaaaacgagcttgttggaacagttgcgacaggaagggataacgaggaaacggccagagcactgggccaaacgcatgggaaacaaaaatattaacgtaaaaacctctcgaggcacaaatatgtcaacagttggggtagatatcggcgattgggtgtatgaaaaaaaagtccgtaatcaatcatcacacggtcctgtaattttcagaacgtgggattttggtggacaaaaagaatattacgccacccatcagtactttttatcaaaacgaagcttatatttggtcgtttggcgaataaccgacggccatcgcggcatcaacgagattttgcaatggcttgttaacatccaagcccgggcaccaaactctcctgtaatcatagtcggcacacactacgacgtggtgcaagaatgtaaccctaacatctcggaagagtaccagcaattaattcgcgacaagtttatcaatattgttgatgcggaaaaatgcggcttgcctcgagttctggacacgattgaaatcagctgcaggactagacacaacgtgaagttgctgtgtaatttaatctacgacaccgtgttcagtttacggccaccaggaagcaaggaattgttgttggaacaaaaagtaccggccacgtacttggcattagaggatgtggttaatcacatcgcggccgaacggcgggtcaatgggcttgacccggtcctgaatgccgaacagtaccggactattgtaacgacggaaatgcagcaacggttcaatcgggtgttcagggactgggctgagctccaccaagccaccttatttttgcacgataatggtgttttgctgcattacgacgatgcaactttgaaagatctctactttttggacccccagtggctgtgcgatatgttagcacatgttgttaccattcgtgagattaatccttttgcgcgctccggtgtcatgaaacttgacgatctcaagcacatttttaaagctagtagtatcggaccaatggatgcgcgtggttatatcgtcaatttgttgaataaattcgaagttgcgctaacttgggattcccgcactttgttaatcccgtcacttctaccctcggaagaggatattctcctagcgcagttataccccggccaatttcaccctttagtcaaggttaaagtgccgctaaggtcacgcggatgggcaatcaggaataaaaaaataactgtttcgccaaaatcggtcttgtaccgggatacgggcgcccagggcaagtttcaaatgagtttgccttcgacttcgtcttcgaaaagtgaaattgaagaggaggcaaagtatcaagtgacgagttcgtcaactgaccaatcaatcacccgactgcttttaatgtcgtacttcccgtcagggttttggtcacgacttatttcacgaattttggccgacgacactattattgatattatacgatcattttttattctcccgaaagaggtggcgcaggatttagatctggtcaagttgctagatttgaaggctgaatgggtgctttggcaaaccggccttcagctgaaatacggcgatattacgttattccgaatgagggaaattttgcacaactcttcgtcctactaccgacagttgaaatttcgtttgaaacaagacggagtttggtgcgacgtcgacctccagaactcctcaatcctcgaaatccacttcccggtggattctttggtcataaaaccggtcactagcgcgaccacagatttgagcaaatacactttaaagactgatcttgtaattaactgtactccggaatcgacagcccaacttttggctttagccgtcgatcacgtcgatatacttttagaggattggtacccgactctcggtactcgcttcgtacatacgtcggagggcaagtttttagttactcggcttgtgccttgtcctcgctgcttgcagcagtcgagcgatattgacccgaattttcccaacacacctgacgttgcaccaaataaaaattttctcgaagctgtgaaccaaatggacgccgaaggcgaccgttaccgcattcgcaaatcgcaagaatcgtacacttcagagtgtgacagtggtgtcgggccggactcgaccggctcgagccgcatgccctcgatggaaggccatccaggggttcagaccgaagatgttaacgctaataaccagctgtactactcctggatggtcgaggagtgcattctggcgtcgtatggcacgaagtgtgtcgtgtgtcccgcccacggtgacgtgtccttggcccggatagcgcctgatacgatttttttggacttgggcgagcgttacatcatccgttcagagaacgtaaaacgcggccggttattaggccgtggtgcgtttggtttcgtgttcaaaggcacgtgcaaggtaagaggtagcaacacaatgatcgacgttgcaatgaaaatgttgcaaccggtgccgcccggccccaatgcccgccaatcggcgattgtggcctacaaagcggcccaaggcaaatgggaccgcgatcccctccaatacgcttgtaaagcctactgtaccgcccggcaagagctcaacattttgctgtctttgagacacccgaacattgttccctttgttggcgtttgcaccagtccgctagccatagtcctggaccttgcaccacaaggtgccttggacctggtcttgaggcattatagacgctctggcgcgaaagtggggccttatacgttacaagcgatcattttacaagttgccaaagcaatcgagtatttgcaccgccaacacatcatttaccgcgatttaaagtcggaaaatgtgctggtttgggaaatgccgccgccttttgtcgaccaccccgaccatccggtgcacatcaaagtggccgattatggcataagtcggctcactttgccgtcgggtgcgaagggttttgggggtactgagggtttcatggctcccgaaattatgcggtacaacggggaggaggaatataccgagaaagttgattgtttctcattcggaatgtttatttatgaattattgacgttgcatcaaccgtttgagggtcacgagtcggtcaaggagtgtattctcgaaggtgggcgtccgccgttgacttacagggagacgttatatccgagttatttcctagatttgatggtcttgtgttggtcacaacaaccgaaagatcggccatcagccagtcaaattgtctccatagctagcgcccccgaatttacccacttgagcgatgttacctcgcttaagcacgaggcttcggttgtggcgactgccagcgcagcagtgacccatataacagatgatggtttgtcgggttctgaaatgtggctaatgtgttccaattctcgaattgacttgttgctggcggctgaccgcgggtggctccaataccacaccatggccttgcctatccgtgccacagcggcttgtaccgtcgcaaatgctgtgtggatcggcgactattccggcaatcttcacgcctattcggccagtgacggctaccgcctcttctcatacgccctcgaaaccgagccaaacacacaagttgaggcactgttgtatcttccaccgttgaaaagggttgcttgtgccctttctaacggccggctgtttctagtcaactctgaagcggtgcctacgacaccgacagccgctgaggggacgtttgtaatgacagagctcggttccaattcggttattaattgtttgtgtgcgatttttaaagacggtggagcggtttgtgaattgtggtgtggtgaatccaatggtcaaatctccatttttataattaaagaccacgttgttgccggtcgcgaaaccatgaaccactaccagcccattattgagcaagtaaatgttatgaatttgatagctgccgataaccacgtctggtcttacgtgcgccctggctgtattatttaccaatgggaccaaaaaaatcggactattatcaataaacttgattgttcgaaacttgtaccgtgttccgaaagtcttaaaagtattgcgattgaagagcatttaagtccgacgaattgtcaagttactagtttggctgtgttgaacgaagaactgtacattgggacaacctggggttgtgttataatagcagagcgagctacgctaaggccgattacgattttcaggccgtatgaagaagaagtgaaagcgatagtaccgttagcaatgtgtaaaagtattaacgaaagtcatgataatacgccgttgattgccacgattggtcgaggttacagaaatctgttgtctcggtatacagacgttccagtcagcactggacactctttacagagtccgatgtcgaatgctccagtggtggccaataagcaaaatatgttcgttttgttgtggcgagctgaacactggaatgctgtctaa
Tribolium Transcript: 3' UTR:
atgctttttcattccatggatcttgagtgataatttaatacctaagtaataagttgtaactgtctttttgtgatttttattacaaattaggttttattttgtatttttatttatattttaagctgtgattctctcagtcattttaacttgttacaaaaacttttattgaataaaatgttacgatgtt
Tribolium Protein:
MLDSSPEDEDFPGRLLHQAALWDNTHLLMDLLEGGQQAFLNSQDSWGRTPLHAAAINDNSGCLTILLNAGADPNIRCGPRGDQKIPLHLSAEHGHNSNVSILLQHDSDIQILDANGMSALDLAEKAGHVKCVTLLKEAVDAREKARLDIHAALRDACCQGDVTTVSSLLASLSKDAELIVNMAPSGASTLLFTACQTGCKDIVKVLLDHGADGRYHPVTKYSPLYIACHYGHRDIVEMLLLRFPELIQQHTVERWLPIHASAINGHVDVLELLFHFPYPSHVLRKFRDGSEQWEYFMPFDINERDATGQNILYMACLVGNKKLLDVILKFKVKATRIQADDEVTPVSESSVVLSPTKRRISDGIQSILSKLNLSRDNSSESDVDPNSSMICPLRIDMYCNNNTETALHAAIKGKHYDIALALLNAGANSNAVIKAYHDLENLSCCSVEEDSNYGQSTGLVEACRNRDVPIVDLLLKYGARDDECKALSVAVQNKDETLTAKLLSTKAHPDPEFKINKKAMTENVNSSQFTIFSNVTNLTYSALFPNTPTMINWHNQRCNLTQIRTQWLTDAALHLNQKLKQNPRSYDIALYAITRLDVSNNNLTSIPLAVFQLYSLRYLNIGQNKIEKLPTPVASPTKKGVRKKNANEELNYSCPVLEELYLQDNRLDHIPDAIFRLPSLVTLVVSNNKLQQLSYNMWLAPKLKELNASFNLLKELPTNMSETQEECDKMSVSSSDSQLSSHCDHDSESEVRCDFDLDSKYLSSDENSQCVKYRKKERSFIQFDLAKHHIWSKNVEVTEQILHTDETVTEQSSKLSALNLAHNLFTSIPVVLPCLAVNLTRLNMAFNSLRSMSHITSYPSSLKQLDLSHNQITCWPSLPQVEAQDVMELANIACYASSVGSREKVSGIAARRQPGRSVRNTVLHSVCSHRRHLKLDSLRTLILADNQLSRIQLSTDDDGDVSAVEEDDLERRKEINTHFEDDFICVSFSKVVGNPAAGKARLMFPNLSMLDVSNNNLKEIPININELSNLSVLNISGNLDVNELPPQMGLLSRLWNLNTRGCSLQDPLKSMIDSKKYKTMDIVGYLKSVLEDARPYARMKLMIVGVQGIGKTSLLEQLRQEGITRKRPEHWAKRMGNKNINVKTSRGTNMSTVGVDIGDWVYEKKVRNQSSHGPVIFRTWDFGGQKEYYATHQYFLSKRSLYLVVWRITDGHRGINEILQWLVNIQARAPNSPVIIVGTHYDVVQECNPNISEEYQQLIRDKFINIVDAEKCGLPRVLDTIEISCRTRHNVKLLCNLIYDTVFSLRPPGSKELLLEQKVPATYLALEDVVNHIAAERRVNGLDPVLNAEQYRTIVTTEMQQRFNRVFRDWAELHQATLFLHDNGVLLHYDDATLKDLYFLDPQWLCDMLAHVVTIREINPFARSGVMKLDDLKHIFKASSIGPMDARGYIVNLLNKFEVALTWDSRTLLIPSLLPSEEDILLAQLYPGQFHPLVKVKVPLRSRGWAIRNKKITVSPKSVLYRDTGAQGKFQMSLPSTSSSKSEEIEEEAKYQVTSSSTDQSITRLLLMSYFPSGFWSRLISRILADDTIIDIIRSFFILPKEVAQDLDLVKLLDLKAEWVLWQTGLQLKYGDITLFRMREILHNSSSYYRQLKFRLKQDGVWCDVDLQNSSILEIHFPVDSLVIKPVTSATTDLSKYTLKTDLVINCTPESTAQLLALAVDHVDILLEDWYPTLGTRFVHTSEGKFLVTRLVPCPRCLQQSSDIDPNFPNTPDVAPNKNFLEAVNQMDAEGDRYRIRKSQESYTSECDSGVGPDSTGSSRMPSMEGHPGVQTEDVNANNQLYYSWMVEECILASYGTKCVVCPAHGDVSLARIAPDTIFLDLGERYIIRSENVKRGRLLGRGAFGFVFKGTCKVRGSNTMIDVAMKMLQPVPPGPNARQSAIVAYKAAQGKWDRDPLQYACKAYCTARQELNILLSLRHPNIVPFVGVCTSPLAIVLDLAPQGALDLVLRHYRRSGAKVGPYTLQAIILQVAKAIEYLHRQHIIYRDLKSENVLVWEMPPPFVDHPDHPVHIKVADYGISRLTLPSGAKGFGGTEGFMAPEIMRYNGEEEYTEKVDCFSFGMFIYELLTLHQPFEGHESVKECILEGGRPPLTYRETLYPSYFLDLMVLCWSQQPKDRPSASQIVSIASAPEFTHLSDVTSLKHEASVVATASAAVTHITDDGLSGSEMWLMCSNSRIDLLLAADRGWLQYHTMALPIRATAACTVANAVWIGDYSGNLHAYSASDGYRLFSYALETEPNTQVEALLYLPPLKRVACALSNGRLFLVNSEAVPTTPTAAEGTFVMTELGSNSVINCLCAIFKDGGAVCELWCGESNGQISIFIIKDHVVAGRETMNHYQPIIEQVNVMNLIAADNHVWSYVRPGCIIYQWDQKNRTIINKLDCSKLVPCSESLKSIAIEEHLSPTNCQVTSLAVLNEELYIGTTWGCVIIAERATLRPITIFRPYEEEVKAIVPLAMCKSINESHDNTPLIATIGRGYRNLLSRYTDVPVSTGHSLQSPMSNAPVVANKQNMFVLLWRAEHWNAV
Forward Primer 1:
5’- AACGTCCATACCACTGGCTG
Reverse Primer 1:
5’- ACCGAAATATAGCGTCCGGG
Forward Primer 2:
5’- TTGGGCGAGCGTTACATCAT
Reverse Primer 2:
5'- AGGACTATGGCTAGCGGACT
Gel Picture:
Cloned Fragment:
ttgggcgagcgttacatcatccgttcagagaacgtaaaacgcggccggttattaggccgtggtgcgtttggtttcgtgttcaaaggcacgtgcaaggtaagaggtagcaacacaatgatcgacgttgcaatgaaaatgttgcaaccggtgccgcccggccccaatgcccgccaatcggcgattgtggcctacaaagcggcccaaggcaaatgggaccgcgatcccctccaatacgcttgtaaagcctactgtaccgcccggcaagagctcaacattttgctgtctttgagacacccgaacattgttccctttgttggcgtttgcaccagtccgctagccatagtcct
Injection concentration:
2 micrograms/microliter
Number Injected:
15
Number Survived to Adult:
12
Number w/ RNAi Phenotype:
0
Percent Penetrance:
0
Assay Description:
We imaged the beetles, their heads, and antennae under high resolution magnification and observed no distinction from the wild type.
RNAi Phenotype Description:
No RNAi phenotype was observed
RNAi Phenotype/Assay Images:
Discussion:
There was no distinction found between the LRRK RNAi Tribolium and the wild type even on their antennae (where past studies had observed a distinction in Drosophila). If everything was successful, as it appeared to be, this denotes one of two results. First, our results may be due to a difference between the Drosophila neurite, which develops from the cell membrane (and has been shown to increase in density with the knock down of LRRK), and Tribolium’s multicellular bristle which we observed to be seemingly unchanged. Alternatively, this may indicate that LRRK neurite growth regulation is not conserved in Tribolium. These hypotheses could be tested through performing in situ hybridization of LRRK to GFP to track its expression. MRI could be performed on beetles to observe their neurite brain growth. Further studies could be performed on the embryonic tissue development, especially regarding the cytoskeleton of neurites.
However, there are multiple procedural errors that could have been responsible for our failure to visualize a distinct mutant phenotype. No distinct phenotype may have been observed because the RNAi didn’t take. This could be due to an injection concentration that was simply too low to have a significant effect, or a sequence match issue. qPCR was not utilized, but could be performed in future studies to ensure that LRRK was successfully knocked down. It is also possible that the gene functions in development prior to the larval stage, which is when our beetles underwent injection. If this was the case, parental RNAi could be utilized to better describe the LRRK mutant phenotype in Tribolium. Furthermore, we were only able to utilize one method of observation to assess the phenotype of our beetles due to time constraints. Thus, it could be that there was a mutant phenotype present, but our method of assessment was insufficient in quantifying it. Higher magnification, such as scanning electron microscopy, may be required to observe phenotypic distinctions.
Notebook:
Annotated Genbank File:
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