DOI: 10.6084/m9.figshare.27926931.v1
title: PRMT5 isoforms during zebrafish developmental stages

description: This file contains information about mass spectrometry data for the manuscript entitled “Protein Arginine Methyltransferase 5 Governs Expression of Early Cardiomyocyte Markers during Zebrafish Development." 1) peptide and protein identifications based on mass spectrometry; 2) a collection of protein sequences, peptide sequences (with modifications), and structures for capturing the scores associated with ranked peptide matches for each spectrum searched. The goal of the mass spectrometry analysis was to confirm or reject identification of protein arginine methyltransferase 5 (Prmt5). Isoforms of Prtmt5 were detected by immunoblotting. Mass spectrometry was used to confirm that the protein identified by immunoblotting was indeed Prmt5.<b>Prmt5 expression fluctuates during zebrafish development.</b>To investigate the role of Prmt5 in zebrafish development, we sought to determine its mRNA and protein expression during early stages of zebrafish embryogenesis. Western blot analysis revealed that Prmt5 protein expression can be detected as early as 6 hrs post-fertilization (hpf), gradually increases 9.3-fold by 18 hpf, and decreases 2 to 3-fold between 24 and 72 hpf.Similarly, real-time RT-PCR analysis at the same developmental stages showed that prmt5 mRNA can be detected 1 hpf and its levels increase slightly by 18 to 72 hpf. Remarkably, despite the lack of any significant change in prmt5 mRNA levels between 18 and 72 hpf, Prmt5 protein expression showed dynamic changes at 18 and 72 hpf, which were accompanied by a change in mobility of Prmt5 protein at 72 hpf compared to other developmental stages.To gain further insight into the unique pattern of Prmt5 protein expression and to understand the shift in its size, we used mass spectrometry to analyze the two Prmt5 isoforms, which were detected at 24 and 72 hpf. Matrix-assisted laser-desorption ionization (MALDI) mass spectrometry was performed for detection of PRTM5 (Ultraflextreme, Bruker Daltonics, Bremen, Germany). Mass spectra were collected in the range from 500 to 3,500 m/z values, positive mode, single protonated, and with a collection of a minimum of 10,000 laser shots for every single spectrum by using FlexControl software (Bruker Daltonics, Bremen, Germany). Tryptic autodigestion peptides 842.51, 1045.56, and 2211.10 Da were used for internal caibration. Analysis of spectra was performed with FlexAnalysis software (Bruker Daltonics, Bremen, Germany). Collected mass lists were used for identification by using ProFound and Mascot search engines (http://prowl.rockefeller.edu/prowl-cgi/profound.exe and https://www.matrixscience.com), with mass tolerance +/-0.1 Da, and no restriction of species, pI, and molecular mass. For identification, the number of matched peptides and coverage of the identified protein with detected peptides were also considered. NCBI database (version of 2019) was used in the searches. The significance of identification was set to p&lt;0.5, one missed cleavage was allowed, and the expectation value was set to &gt;0.95. Identified PRTM5 peptides were matched and assigned to PTRM5 peptides generated by in silico digestion with trypsin as described in the maniuscript.We found unique peptides present in Prmt5 protein isolated at 24 hpf, but not in Prmt5 protein isolated at 72 hpf. Amino acid sequence alignment of the four available zebrafish Prmt5 variants indicated that all of the identified peptides were present only in Prmt5 variant 1 (Accesssion no. Q503E3). Mobility of Prmt5 at 72 hpf indicated a lower apparent molecular weight compared to the 24 hpf zebrafish Prmt5 variant 1. In addition, the predicted molecular weights of the already identified zebrafish cDNAs varied in size between 69.41 kDa for variant 1 (Accesssion no. Q503E3), 70.73 kDa for variant 2 (Accesssion no. A0A8M6Z3C8), 72.71 kDa for variant 3 (Accesssion no. A0A8M9PS04), and 85.36 kDa for variant 4 (Accesssion no. Q68EH4). Taken together, these results suggest that the Prmt5 protein expressed at 72 hpf corresponds to a novel splice variant of a smaller size with a predicted molecular weight lower than 69.41 kDa, and which lacks the 6 peptides identified by mass spectrometry.Table 1. Prmt5 peptide sequences expressed at 24 but not at 72 hpf.Peptide sequences Residues Position Computed Mass1- LSPWIETDSELTTERR 82-97 1931.9582- WLGEPIKAAILPTSIFLTNK 217-236 2211.2663- HSEKDLR 268-274 883.4514- EWTSPEK 418-424 875.4025- ADIIVSELLGSFGDNELSPECLDGAQHFLK 425-454 3216.5636- EVTLSIKPETHSPGMFSWFPILFPLK 556-581 3000.581Total computed mass of peptides in the Prmt5 isoform observed at 24 hpf. 12960.69<br>The total computed mass of 12,960.69 Da, assigned to extra peptides in Prmt5 variant 1 (69.41 kDa, accesssion no. Q503E3) present at 24 hpf, is missing in the shorter version of Prmt5 variant found at 72 hpf.