SMIM19

SMIM19, also known as Small Integral Membrane Protein 19, encodes the SMIM19 protein. SMIM19 is a confirmed single-pass transmembrane protein passing from outside to inside, 5' to 3' respectively. SMIM19 has ubiquitously high to medium expression with among varied tissues or organs. The validated function of SMIM19 remains under review because of on sub-cellular localization uncertainty. However, all linked proteins research to interact with SMIM19 are associated with the endoplasmic reticulum (ER), presuming SMIM19 ER association

Gene SMIM19 is also commonly known and referenced as C8orf40 (chromosome 8 open reading frame 40). SMIM19 also has a few other lesser known names, such as LOC114926, Doyzeeby, and Beeveybu. The SMIM19 gene is located on the plus strand at 8p11.21 in humans. The SMIM19 gene is composed of 4 total exons and spanning 14.04 kb from 42,541,155 bp to 42,555,193 bp. The upstream neighboring gene to SMIM19 is SLC20A2.

Transcripts

Variants There are four validate transcript variants for SMIM19 that all encode the same protein, as they only differ in sequence within the 5' UTR. Transcript variant 1 represents the longest transcript +Transcript Variants of SMIM19Transcript VariantRefSeq AccessionLengthDescriptionNumber of ExonsTranscript Variant 1NM_001135674.23704 bpEcodes Isoform 14Transcript Variant 2NM_001135675.23486 bpEcodes Isoform 1; Lacks portion of 5' UTR4Transcript Variant 3NM_138436.43036 bpEcodes Isoform 1; Lacks portion of 5' UTR4Transcript Variant 4NM_001135676.22967 bpEcodes Isoform 1; Lacks portion of 5' UTR4

Features The SMIM19 mRNA is composed of one major polyadenylation sequence and site combination and three alternative ones, along with an upstream in-frame stop codon. Not all variants contain the upstream in-frame stop codon as it is present in the portion of the 5' UTR that varies. The polyadenylation sequences and site are consistent among all transcript variants.

Protein

Isoforms There is only one validated SMIM19 protein isoform that is encoded by all four transcript variants. It is 107 amino acids long with a molecular weight of 12.44 kDa. The isoelectric point varies among SMIM19 organism and shows a pattern based on taxonomical group, likely due to post-modification variations between taxonomical group +Isoelectric Point Taxonomical PatternOrganismTaxonomical GroupIsoelectric Point (pH)Homo sapiensMammal5.3Mus MusculusMammal5.62Danio rerioFish5.78Xenopus tropicalisAmphibian6.08Trachemys scripta elegansReptile7.97Gopherus evgoodeiReptile7.97Gallus gallusBird9.34Taeniopygia guttataBird9

Amino Acid Composition The SMIM19 protein contains a validated transmembrane region. There is no indication that SMIM19 is rich or poor in any amino acid; no amino acid or combination of amino acids were outside of the standard deviation in relation to abundance. There was no significance detected with the spacing of cysteines as SMIM19 contains none. Similarly, there were no repetitive structures i.e., separated, tandem or periodic repeats, found in SMIM19 protein sequence

Transmembrane/ Hydrophobic Region The SMIM19 protein contains a transmembrane region, which is also considered a hydrophobic region that spans 19 amino acids. The right a TMpred output predicted orientation analysis of the SMIM19 protein demonstrates that the protein is likely oriented outside to inside, which was used to structure the TOPO2 diagram.

Positive Amino Acid Run Consistent with the majority of SMIM19 homologs, there is a positive amino acid run almost immediately following the transmembrane region: KRR (Lysine, Arginine, Arginine).

Motifs There were four matches for motifs with the SMIM19 sequence that are predicted to be significant. Casein kinase 2 can play a role in cell cycle regulation, DNA repair, and necessary for cell survival; down-regulation of Casein kinase 2 can promote tumorigenesis. Protein kinase C is a protein-regulator and is highly involved in various signaling cascades. Dendritic Cells- Specific Transmembrane Protein (DC-STAMP) is a seven- pass transmembrane protein specifically found in dendritic cells, often associated with immunological functions. Based on Eukaryotic Linear Motif predictions, there are strong sequence matches for cleavage site motifs following the transmembrane region, which also remain consistent among orthologs

+Motif Sequence Matches with SMIM19 ProteinMotif NameAbbreviationAmino Acid PositionAmino Acid SequenceCasein kinase II phosphorylation siteCK2_PHOSPHO_SITE17-20TVHECasein kinase II phosphorylation siteCK2_PHOSPHO_SITE62-65TVHEProtein kinase C phosphorylation sitePKC_PHOSPHO_SITE87-89SRKDC-STAMP-like proteinDC_STAMP31-54IVILVSFGLFMYAKRNNKRRIMRIF

Secondary Structure

Based on ALI2D and Phyr2 (diagram pictured) data, it is determined that an alpha helix is likely present at the beginning of the sequence right before the transmembrane sequence with high confidence analysis. The transmembrane region structure following this first alpha helix, varies in structure per program used for analysis, so no conclusions could be made. The second beta sheet is consistent amongst various programs, and is likely a strong candidate for prediction. Following this, the large alpha helix predicted remains fairly consistent through all orthologs with high confidence from both Pyre2 and ALI2D. The end beta sheet is consistent between program analyses and predictions but remains in low confidence, so no definitive conclusions could be made.

Tertiary Structure

I-Tasser results of both the SMIM19 upstream and down stream sequence to the transmembrane domain (pictured left and right respectively) were analyzed with iCn3D. The SMIM19 protein is analyzed as a whole cause the cytosolic and extracellular sequence flanking the transmembrane domain attempt to coil together, producing a false predicted 3D structure as they should never interact being there is a membrane between them. Therefore, each upstream and downstream sequences was analyzed separately. Within each diagram there is a yellow highlighted section where the transmembrane sequence would meet each respective sequence. No strong conclusions can be made about SMIM19 tertiary structure beyond a large alpha helix present in the downstream sequence to the transmembrane region, consistent with Ali2D and Phyr2 results above

Regulation and Expression

Gene-level Regulation

Promoter The most conserved promoter among SMIM19 orthologs was GXP_9002686 on the positive strand spanning 1962 bp, located between 42,540,128 and 42,542,089. It is also supported by the greatest number of transcripts. +Unique Promoter Results for SMIM19Promoter IDSize (bp)StartEndStrandNumber of TranscriptsGXP_900268619624254012842542089 +10GXP_205977710474254014842541194 +1GXP_952656710404254260542543644 +0GXP_952656810404254424842545287 +0GXP_321248112064254526242546467 +1GXP_952656910404254736442548403 +0

Transcription factors The below selected transcription factors were chosen based on conservation among species first, and then further parsed for high matrix similarity and high number of proposed binding sites with in an extended SMIM19 promoter region. Conservation was highest further that desired from transcription start site. +SMIM19 Transcription FactorsTranscription factorDescriptionStrandMatrix similaritySequenceFKHDFork head domain factors +1caaaaaaAACAaaacaaFKHDFork head domain factors +1caaaaaaAACAaaacaaFKHDFork head domain factors +0.999gcccggcAAACaatcagPIT1GHF-1 pituitary specific POU domain transcription factor +0.953tatataaatACATataaatHOMFHomeodomain transcription factor +0.995gtgagttTAATtgtaacagCARTCart-1 (catrilage homeoprotein 1) +0.995gagttTAATtgtaacagatgaHBOXHomeobx transcription factors +0.944gacttatAATTaccagtcaDLXFDisral-less homeodomain transcription factors -0.989gctgactggtAATTataagHOXFParalog hox genes 1-8 from the four hox clusters A, B, C, D +0.985acttctaATTAccagtcagLHXFLim homeodomain factors -0.979tatacatttTGATtaagttctctCAATCCAAT binding factors +0.926ccagCCACtgacatcOTC1Octamer binding protein +0.992cctATGCaaattcatBRNFBm Pou domain factors -0.982cttgacctaagTAATgaatCARTCart-1 (catrilage homeoprotein 1) -0.995ttattTAATtgtgtagtgactARIDAT rich interactive domain factor +0.985taaaAATAcccaaaagggactFKHDFork head domain factors +1ttttgaaAACActacggNR2FNuclear receptor subfamily 2 factors +0.904cctggtgggaCAATgtacacgacccNKXHNkx homeodomain factors +0.986cagcgTGAGtgbccccgcgMYBLCellular and Viral myb-like transcriptional regulators -0.957gggccgccgCAACtggcccgtETSFHuman and murine ETS1 factors -0.991ctctcccaGGAAgcagcccgg

Expression Patterns According to RNA-seq data from Human Protein Atlas, SMIM19 has ubiquitously medium to high expression in all tissues with low specificity. Comparatively, there is higher expression in liver, muscle, some glandular tissue, and various immune cells. Expression in the brain is comparatively consistently lower.Chromatin Immunoprecipitation (ChIP) data for SMIM19 in mice even more confidently display ubiquitously medium to high expression in tissue; No tissue seems to fall below the 50 percentile rank for expression. Muscle tissue has high expression in mice as well. Adipose tissue and diaphragm tissue uniquely are high in expression comparatively.

in situ hybridization data of SMIM19 expression in sagittal sectioning of whole embryos produced no definitive conclusions. No significantly abnormal cellular expression were observed.  SMIM19 appears to be uniformly expressed.

Transcript-level Regulation

miRNA binding sites Hsa-miR-1206 and hsa-miR-433-3p were both highly ranked microRNAs in reference to predicted sequence matching with SMIM19 transcript. Each position and predicted sequence pairing is displayed in the image below.

mRNA-binding proteins The tables below represent the most significant RNA-binding proteins based on relevancy and match score to the SMIM19 5’ UTR and 3’ UTR. Selection was not based on conserved sequence observed between orthologs being there is little conservation of the SMIM19 5’ and 3’ UTR outside of mammals. +SMIM19 5' UTR Selected RNA-binding Protein PredictionsRBP NameFull NameScoreRelative ScoreTranscript PositionMatching SequenceSummarySFRS1splicing factor, arginine/serine-rich 110.87100%413-419ACGCGCAProtein can activate or repress splicing; regulator of splicingFUSfused in sarcoma7.37100%771-774GGUGPart of a complex involved on pre-mRNA splicing and export of mRNA to the cytoplasmEIF4Beukaryotic translation factor 4B8.0588%431-437GCGGAAAProtein required for binding og mRNA to ribosomessap-49spliceosome associated protein7.5686%120-125GCGUGAInvolved in various pre-mRNA splicing complexesZRANB2zinc finger, RAN-binding domain containing 28.3981%27-32CGGUAAProtein is a splicing factor required for alternative splicing of specific transcripts +SMIM19 3' UTR Selected RNA-binding Protein PredictionsRBP NameFull NameScoreRelative ScoreTranscript PositionMatching SequenceSummarySNRPAsmall nuclear ribonucleoprotein polypeptide A11.95100%1995-2001AUUGCACAssociates with a protein to bind 5' splice site of precursor mRNAs; required for splicingNONOnon-POU domain containing, octomer-binding8.95100%1491-1495AGGGAPlays a role in transcriptional regulation and RNA splicingPABPC1poly(A) binding protein, cytoplasmic 18.72100%1388-1392AAAAAProtein shuttles between nucleus and cytoplasm and binds 3' poly(A) tail of eukaryotic messenger RNARBMY1A1RNA binding motif protein, Y-linked, family 1, member A18.67100%2149-2153CUCAAFunctions as a splicing regulatora2bp1Fox-1 homolog A (Ataxin 2-binding protein 1)8.65100%906-910GCAUGRegulates tissue-specific alternative splicing

Secondary structure

Large variation in SMIM19 5' UTR between variants within Homo sapiens and orthologs, makes secondary structure of possible regulation site fairly unreliable.