Dataset Information

MCherry contains a fluorescent protein isoform that interferes with its reporter function

ABSTRACT: Fluorescent proteins are essential reporters in cell biology and molecular biology. Here, we reveal that red-fluorescent proteins possess an alternative translation initiation site that produces a short functional protein isoform. The short isoform creates significant background fluorescence that biases the outcome of expression studies. Our investigation identifies the short protein isoform, traces its origin, and determines the extent of the issue within the family of red fluorescent protein. Our analysis shows that the short isoform defect of the red fluorescent protein family may affect the interpretation of many published studies. Finally, we provide a re-engineered mCherry variant that lacks background expression as an improved tool for imaging and protein expression studies.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Escherichia Coli

SUBMITTER: Davi Fonseca

LAB HEAD: Martin Hohmann-Marriott

PROVIDER: PXD032954 | Pride | 2022-04-19

REPOSITORIES: Pride

ACCESS DATA

Similar Datasets

Project description:The most common genetic causes of steroid-resistant nephrotic syndrome (SRNS) are mutations in the NPHS2 gene, which encodes the lipid-binding protein podocin. Mass spectrometry and cDNA sequencing revealed the existence of a second shorter isoform in the human kidney in addition to the well-studied canonical full-length protein. Distinct subcellular localization of the shorter isoform that lacks part of the conserved PHB domain suggested a physiological role. Here we analyzed whether this protein can substitute for the canonical full-length protein. The short isoform of podocin is not found in other organisms except humans. We therefore now analysed a mouse line expressing the equivalent podocin isoform (podocinΔexon5) by CRISPR/Cas-mediated genome editing. We characterized the phenotype of these mice expressing podocinΔexon5 and used targeted mass spectrometry and qPCR to compare protein and RNA levels of podocinwildtype and podocinΔexon. After immunolabeling slit diaphragm components, STED microscopy was applied to visualize alterations of the podocytes’ foot process morphology. Mice homozygous for podocinΔexon5 were born heavily albuminuric and did not survive past the first 24 hours after birth. Targeted mass spectrometry revealed massively decreased protein levels of podocinΔexon5, whereas RNA abundance was not different from the canonical form of podocin. STED microscopy revealed the complete absence of podocin at the podocytes’ slit diaphragm and severe morphological alterations of podocyte foot processes. Mice heterozygous for podocinΔexon5 were phenotypically and morphologically unaffected despite decreased podocin and nephrin protein levels.The murine equivalent to the human short isoform of podocin cannot stabilize the lipid-protein complex at the podocyte slit diaphragm. Reduction of podocin levels at the site of the slit diaphragm complex has a detrimental effect on podocyte function and morphology. It is associated with decreased protein abundance of nephrin, the central component of the filtration-slit forming slit diaphragm protein complex.

Dataset Information

MCherry contains a fluorescent protein isoform that interferes with its reporter function

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure