Table 1.
PCR primer and cycling conditions employed to identify transcripts in human and mouse.
Fig 1.
Organization of the PKD1 locus and transcripts expression profile from GTEx.
A. Workflow of analysis of public databases to generate a heat map of PKD1 transcripts. From GTEx 40 PKD1 transcripts were identified from 53 body sites. The median expression levels for each tissue site were evaluated and normalized. Heatmaps for transcript expression was generated via Morpheus software package (https://software.broadinstitute.org/morpheus). B. The median expression levels (in TPM) of highly expressed transcripts (normalized by column maximum) were represented in heatmap of Ensembl Transcript (ENST). PKD1-208 stands out as the most abundant and highly ubiquitous transcript. Full-length PKD1 transcripts are highlighted in yellow and red. Heat map shows the color scale relative to transcript number. C. Genomic track of human PKD1 obtained from Ensembl (GRCh38.p12), representing major transcripts. Transcripts in red, are transcripts predicted to be protein coding. PKD1-208 is located on the 3’ end of the HmPKD1 locus and is predicted to be a processed transcript.
Fig 2.
Sequence and detection of human PKD1-208 alternative transcript.
A. Nucleotide sequence of wild type PKD1 transcript, the red boxed nucleotides delineate exon 42 which is deleted in alternative PKD1 transcript (PKD1-208). Bold sequences are primer sites. Each exon has been color coded. B. Pairwise alignment of nucleotide region for alternative PKD1 transcript and wild type PKD1, showing missing exon 42 in the alternative transcript. C. Schematic diagram of intronic and exon sequences from full-length HmPKD1 starting from intron 40. PKD1-208 transcript has an exon 42 skipping (triangle below exon). PCR primers used in this study are mapped to the diagram (nucleotide sequences are found in Table 1). D. Detection of wild type and alternative PKD1 transcript in human kidney tissue using common PKD1 primers (Listed on the left of the agarose gel image) (lane 1) and specific primers (lane 2 and 3) for the alternative transcript. M, 1Kb DNA ladder. E. Sequence of PKD1-208 cDNA with same color code as in A. Underlined nucleotide sequences represent protein coding region of alternative PKD1 transcript. Bold sequence CTG are potential Leucine translation start sites.
Fig 3.
Identification of a murine alternative transcript within the Pkd1 locus.
A. Transcriptional activity of the human (left) and mouse (right) 3’ end PKD1/Pkd1 locus from UCSC Browser (exons 39 to 3’UTR). This human region has high CpG density (green box). Alignment of ChIP-Seq binding pattern data of selected chromatin marks show strong chromatin activity in various human cell lines (NHEK, HUVEC, K562, NHLF, H1-hESC, GM128878) but weak in the mouse newborn kidney genome. Mouse intron 39 to exon41 contains two candidate cis-regulatory elements (CRE) marks indicative of promoter-like biochemical signature from H3K4Me3 (pink box). Mouse exon 46 and 3’end UTR contain two proximal enhancer-like signatures (orange box). B. Schematic diagram of mouse 3’ end Pkd1 locus from intron 40 to 3’UTR with corresponding exon size length, with or without exon 42 (red triangle). PCR primers used are mapped over this genomic region. C. Detection of corresponding human alternative transcript in total adult mouse kidney cDNA of endogenous wild-type C57BL/6J (WT) yield Pkd1 band of 420 bp and alternative transcript band of 245 bp (primers 11.06-97.26), and no band in non-template control (NTC) (Left and right panels) and transgenic kidneys Pkd1WT (right panel). Marker: 1Kb Plus DNA ladder. D. Expression of alternative transcript was monitored in newborn kidneys of endogenous WT, transgenic kidneys Pkd1WT and SBPkd1 with same primers as in C. E. Detection of alternative transcript in total adult mouse kidney cDNA with another set of primers (11.06-11.07). Endogenous WT and transgenic kidneys SBPkd1 yield 2 bands at 778 and 603 bp for Pkd1 and alternative transcript bands, respectively. F. Detection of alternative transcript over exon 41-43 junction in adult transgenic SBPkd1 produced the predicted single band of 499 bp (primers 23.01-11.07) at different intensity according to cDNA quantity. G. Detection of alternative transcript over exon 41-43 junction in two newborn WT and transgenic Pkd1WT kidneys (primers 23.02-97.26) was shown by a single band of 148 bp.
Fig 4.
Characterization of the protein encoded by the alternative transcript: Eliosin.
A. Immunoblot detection of a protein derived of the full-length alternative transcript from the cloned cDNA in three reading frames of cTAP A, B or C transfected into HEK293 cells. When in frame, expression of a chimeric protein with the calmodulin binding protein sequence at the carboxy-terminal is detected with a calmodulin specific antibody. Lanes 1, 2 mock transfected control, lanes 3, 4 transfected with cTAP A, lanes 5, 6 with cTAP B, lanes 7, 8 with cTAP C. In comparison to the mock transfected cells, a 48 kDa CBP-tagged protein is uniquely detected (red arrow) in lanes 3 and 4 from HEK293 cells transfected with cTAP A. B. Analysis of the protein initiation site was performed on the cDNA of the alternative transcript with the native 5’ UTR and ORF in pENTR CMV-TO-IRES GFP plasmid (Lane 2) and in two different clones by PCR constructs which include CTG/Leucine initiation site (Lanes 4 and 5) produced ~ 47kDa band. Lane 3 contains an empty plasmid backbone as background control. Plasmid constructs with the first three nucleotides encoding Leucine deleted, making way for Alanine to be the first potential codon (Lane 6), or mutated in the 2nd base of the CTG initiation site, that should convert Leucine to a Glutamine codon (Lane 7), prevented production of the ~ 47 kDa band. The ~ 37kDa (*) band may correspond to another downstream translation start site for this alternative cDNA. Lanes 1 and 8 are empty wells. C. Amino acid sequence of isolated Eliosin with underlined sequence shared with PC1 and with highlighted amino acids representing peptide mapping coverages from proteomic studies. The first five amino terminal amino acids are uniquely expressed in Eliosin. D. 2D Gel Immunoblot analysis on NIH 3T3 cells transfected with cDNA of the full-length alternative transcript (top panel) or control pGreen (bottom panel) were probed with anti-human C-terminal PC1 (NM005) to detect Eliosin. A single spot with an approximate pI of 9.0 and a molecular weight of ~47 kDa is seen in the transfected cells in comparison to control.
Fig 5.
Eliosin is expressed in human cells treated with PC1 siRNA.
Immunoblot of HK2 cells monitored for endogenous Eliosin expression was probed with anti-Polycystin C-terminal NM005 (Top panel). In lanes 1, 2, HK-2 cells were treated with scrambled siRNA or with 3 different siRNAs that bind to sequences located at the 5’ end of the HmPKD1 gene: siRNA-1, lanes 3,4; siRNA-2, lanes 5,6; siRNA-3, lane 7. A band of ~45-47 kDa corresponding to Eliosin and a high molecular weight band likely the full-length PC1 were detected in the scrambled siRNA treated cells (lanes 1, 2). The specific PKD1 siRNAs suppressed full-length PC1 whereas the expected band for Eliosin is unaffected. The ~ 68 kDa band (*) may be an Eliosin heteromer or non-specific background band. Lower image: Same immunoblot from the upper panel probed with anti-actin.
Fig 6.
Eliosin is a component of mitochondria-ER associated membranes.
A. Co-localization studies of mCherry Eliosin transfected in HEK 293 by immunofluorescence with anti-Polycystin1 C-terminus, NM005 (middle panel) and ERMCS/MAM marker IP3R (left panel). Notable co-localization between Eliosin and IP3R is observed. Bar = 10 microns. Pixel size 0.11 µm2, z distance = 0.35 µm per stack. The image is an extended focus image generated by projecting the maximal pixel intensity onto a single plan determined by a ray trace perpendicular to the z stack [44]. B. Immunofluorescence studies on transfected mCherry Eliosin in HEK 293 with the ERMCS/MAM marker Mitofusin 2-GFP show significant co-localization. Bar = 10 microns. Voxel size = 0.28 µm3. The image is an extended focus image. Z distance = 0.28 µm. C. Co-localization studies of SNAP-tagged Eliosin (green, left panel) co-transfected with mCherry dynamin related protein (DRP1) (red, middle panel) by immunofluorescence. DRP1 and Eliosin co-localize in a punctate pattern (arrowheads). Bar = 10 microns, voxel size = 0.28 µm3, Z = distance 0.28 µm. The image is an extended focus image.
Fig 7.
Eliosin can correct the fragmented mitochondria in ADPKD renal cells.
A. Untransfected WT 9−7 cells labeled with Mitotracker green show punctate mitochondria (Top panel). Bar = 10 microns. B-D. WT 9−7 cells labeled with Mitotracker green (B) and transfected with mCherry-Eliosin (red) (C) result in filamentous mitochondria. The merged image (D) shows cells with extended reticular mitochondria. Bar = 10 microns. E. Graph of mitochondria circularity measurements in WT 9−7 untransfected cells labeled with Mitotracker versus WT 9−7 cells transfected with Eliosin or transfected with Eliosin and DRP1 and labeled with Mitotracker. * p < 0.001, ** p < 0.0002.