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RNF128

Ring Finger Protein 128 (RNF128) is an enzyme that's encoded by the RNF128 gene. RNF128 is most known for its link to many types of cancer in the skin, esophagus, and bladder when it's downregulated.[1] [2] The protein for this gene is best known as E3 ubiquitin-protein ligase RNF128. This protein is a type 1 transmembrane protein that functions as an E3 ubiquitin ligase and is also shown to inhibit the activation of IL2 and IL4 cytokine production when expressed in T cells. E3 ubiquitin-protein ligase RNF128 is highly expressed in the liver, adrenal glands, and intestines and also has notable expression in the kidneys, stomach, bladder, and thyroid.[3] This protein lies in the endocytic pathway and contains a signal peptide, a RING zinc-finger motif, a protease associated domain, and a transmembrane domain.

Gene

RNF128 goes by other aliases including Gene Related to Anergy in Lymphocytes protein (GRAIL), E3 ubiquitin-protein ligase RNF128, FLJ23516, and RING finger protein 128.[1]The human RNF128 gene is located at Xq22.3 on the plus strand of the X chromosome and contains 8 exons and 7 introns.[2] The gene is 103,223 base pairs long and spans from 105,937,024 to 106,040,244.[3] This gene also has 234 orthologs in a span of organisms and is conserved in animals such as a dog, cow, mouse, rat, chicken, and a zebrafish.[4] Paralogs for this gene include RNF133, RNF150, RNF148, RNF149, RNF130, RNF13, RNF167, RNF215, and ZNRF4.

[1] Wikigenes(RNF128)- https://www.wikigenes.org/e/gene/e/79589.html

[2] NCBI Gene(RNF128)- https://www.ncbi.nlm.nih.gov/gene/79589

[3] Aceview(RNF128)- https://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/av.cgi?db=human&term=RNF128&submit=Go

[4] NCBI Gene(RNF128)- https://www.ncbi.nlm.nih.gov/gene/79589

Transcript (mRNA)

Isoforms

RNF128 has two reported alternatively spliced transcript variants encoding isoforms. Isoform 1 contains 428 amino acids and isoform 2 contains 422 amino acids.[2] Isoform 1 has a longer transcript. Isoform 2 has an alternative 5' UTR and a different exon one compared to isoform 1. This results in a much shorter N-terminus in isoform 2. Isoform 1 is used more often when analyzing the protein. Isoform 1 includes a signal peptide, protease associated domain, transmembrane domain, and a RING zinc finger domain. Isoform 2 does not contain the same signal peptide or protease associated domain, but does include a similar transmembrane domain and RING zinc finger domain.

Protein

General Properties

The RNF128 gene encodes and type 1 transmembrane protein that has a molecular weight of 46.5 kDa and a pI of 5.92. (Pi Tool- https://web.expasy.org/cgi-bin/compute_pi/pi_tool) This protein functions as a E3 ubiquitin protein ligase that catalyzes Lys-43 and Lys-63 linked polyubiquitin chains and acts as an inhibitor of cytokine gene transcription when expressed in retrovirally transduced T cells.[1] This protein contains 428 amino acids and has two known isoforms. Compared to other proteins, E3 ubiquitin-protein ligase RNF128 has mostly a normal amount of amino acids. One thing that makes this protein unique is that it has more than usual Valines (V+). Valines are V-2-V 7 times, which is an interesting characteristic. One thing to note is that this protein has more negative than positive amino acids as the KRED score is -8. As far as charge distribution goes, there is a positive cluster of 13+ from 232-264 and a negative cluster of 13- from 373-420.[1] The RNF128 protein mouse ortholog has no negative charge clusters and not as many valines as the human ortholog. As mentioned previously, this protein contains a Ring-H2 region, which is a RING zinc finger motif. It also has a transmembrane domain, signal peptide, and protease associated domain. Some highly conserved motifs found were Gen1 and SIMAnti2.[2] There was a unique region found from 232-246. This region was a NLS-BP Bipartite nuclear localization signal.[3] This is interesting because RNF128 is usually found in the cytosol, but it contains a signal for the nucleus.

Secondary and Tertiary Structure

The secondary structure of E3 ubiquitin-protein ligase RNF128 is just about half alpha helices and half beta sheets. It is predicted by many programs that about 85 residues were predicted helix and 117 were predicted to be sheets and the rest either turns or random coils.[1][2] The I-TASSER program showed in confidence that there are about 6 alpha helices and 9 beta sheets within the secondary structure. Tertiary structures of the entire protein, the protease associated domain alone, and the Ring-H2 region alone are shown below. These figures support the data that there are many alpha helicies as well as beta sheets involved in the tertiary structure.

[1] Chou- http://www.biogem.org/tool/chou-fasman/index.php

[2] GOR- https://npsa-prabi.ibcp.fr/cgi-bin/secpred_gor4.pl

[1] SAPS-https://www.ebi.ac.uk/Tools/services/web/toolresult.ebi?jobId=saps-I20200709-160856-0340-12190082-p1m

[2] Elm- http://elm.eu.org/cgimodel.py?fun=smartResult&userId=QiKRohMPjG&EXPECT_CUTOFF=100&r=1&bg=on

[3] MyHits- https://myhits.sib.swiss/cgi-bin/motif_scan

Gene Level Regulation

Promoter

There are two total promoters, but the main promoter (GXP_14319) for RNF128 is 1076 nucleotides long. The transcription start site for RNF128 resides at the very end of the promoter sequence in the last 40 amino acids.

Transcription Binding sites

There are many transcription factors that have a high affinity for binding RNF128's 5' UTR. Some important ones to mention are NFAT, a nuclear factor of activated T cells, EGRF, a Wilms tumor suppressor, and HNF6, a liver enriched cut-homeodomain transcription factor.

Expression

RNF128's expression in human tissues is very specific to the gut. There is very high expression in the liver and fetal liver especially. There is also high expression in the kidneys, adrenal glands, thyroid, small intestines and stomach.

Transcript Level Regulation

There are over 10 stem loops in the 5' untranslated region on RNF128. There are also several areas of the 5' untranslated region that are highly conserved.

Protein Level Regulation

The RNF128 protein contains a signal peptide. This peptide is cleaved at the RGA site 37 amino acids into the protein.[1] Myristoylation sites were predicted giving a significantly positive result when the first 5 amino acids were removed from the sequence.[2] RNF128 also has three predicted palmitoylation sites found using CSS-Palm.[3] Myristoylation and palmitoylation adds a myristoyl and palmitoyl group to the protein which contrasts with N glycosylation because you are adding a hydrophobic tail. These tails burry themselves in the protein to tell the protein is membrane associated. There are 6 predicted O glycosylation sites within this protein.[4] Out of the 6, only one of these O glycosylation sites are above the threshold, so it is assumed that there is only one, although it’s unlikely as RNF128 is secreted.[5] At O glycosylation sites, serines and threonines can be both phosphorylated and glycosylated and under different conditions, can be turned on or off. There are many phosphorylation sites for this protein, most of these being serines and a few threonine and tyrosine.[6] Phosphorylation is on the inside of the cell and can often turn certain signals on or off and can even lead to conformational changes in proteins. There are three significant sites for N glycosylation that are above the threshold.[7] This could possibly protect the protein because of the large sugar complex and attract lectins that bind other proteins. Sugars from this N glycosylation also can change the shape of the protein which also helps it bind to other factors. GPS-Sumo predicted three different sumoylation sites.[8] These sites are similar to ubiquination in that they help target proteins for degradation under the right conditions. This helps the protein get rid of unwanted or needed regions. Research foudn that one third of the time this protein is found in the endoplasmic reticulum, one third in the plasma membrane, and the other third in the golgi.[9] This protein is considered to be localized in the endocytic pathway.

[1] SignalP- http://www.cbs.dtu.dk/cgi-bin/webface2.fcgi?jobid=5F0DD7E600002CE72D6D371A&wait=20

[2] Myristoylator- https://web.expasy.org/cgi-bin/myristoylator/myristoylator.pl

[3] CSS-Palm- http://csspalm.biocuckoo.org/showResult.php

[4] NetOGlyc- http://www.cbs.dtu.dk/cgi-bin/webface2.fcgi?jobid=5F07496D000048E0CEF7DBCA&wait=20

[5] YinOYang- http://www.cbs.dtu.dk/cgi-bin/webface2.fcgi?jobid=5F0DD7FA00002CE7D7515FD9&wait=20

[6] NetPhos- http://www.cbs.dtu.dk/cgi-bin/webface2.fcgi?jobid=5F07487400006C9E3D0E94A1&wait=20

[7] NetNGlyc- http://www.cbs.dtu.dk/cgi-bin/webface2.fcgi?jobid=5F0DD4DB0000662F2F470DB9&wait=20

[8] GPS-SUMO- http://sumosp.biocuckoo.org/showResult.php

[9] Psort-https://psort.hgc.jp/cgi-bin/runpsort.pl

Subcellular localization, signal peptide, cleavage sites, glycosylation, sumoylation, other sites

Homology & Evolution

Paralogs

*Estimated time of divergence, and phylogeny of paralogs

Paralog	E-value	Similarity %	Identitiy %	Relatedness
RNF133	4e-118	58	44	Closely related
RNF150	9e-84	52	38	Mod. related
RNF148	1e-99	49	37	Mod. related
RNF149	2e-76	49	34	Mod. related
RNF130	2e-72	46	33	Mod. related
RNF13	4e-15	37	21	Distantly related
RNF167	5e-14	35	21	Distantly related
RNF215	1e-11	27	18	Distantly related
ZNRF4	8e-10	33	19	Distantly related

Table #: This table gives a list of nine RNF128 paralogs. Percent identity and percent similarity were found using EMBOSS Needle.

Orthologs

RNF128 has 234 orthologs and is conserved in animals such as a dog, cow, ouse, rat, chicken, and a zebrafish.[4] The most closely related orthologs reside in mammals with similarities between 75 and 100 percent. Moderately related orthologs resided in reptiles and birds with similarities between 67-75 percent. Then finally the most distantly related orthologs are amphibians and bony fish with similarity values around 60 percent. Many multiple sequence alignments were run using EMBOSS Global to look at the conservation of amino acids over time. The multiple sequence alignments compared distantly related and closely related homologs of RNF128. Many regions of RNF128 are conserved in the all species from mammals to bony fish, including the protease associated domain, transmembrane region, and the ring-H2 region. The signal peptide is not conserved in any of the more distant homologs, but is conserved in the strict orthologs. The ring-H2 region is the most highly conserved region in these alignments and is conserved in mammals, birds, amphibians, reptiles, and bony fish.

Evolution

RNF128 goes back approximately 433 m.y. The oldest life forms I found the gene to appear in are bony fish like zebrafish. This gene was not found in any invertebrates, fungus, bacteria, etc. The size of the gene family is 2. There are two isoforms produced by alternative splicing for RNF128 and I only found one splice isoform for the most distantly related organism. Figure 1 below shows the divergence of RNF128 overtime and how slowly or quickly it diverged between organisms. When comparing my gene to cytochrome c and fibrinogen alpha, it can be determined that RNF128 diverges moderately slow. I used the slope of each line to find that a 1% change takes 27.7 million years for cytochrome c, 6.9 million years for RNF128, and 2.7 million years for fibrinogen alpha. You could also use the slope of the linear model of RNF128 to estimate the time of divergence for all paralogs.

A phylogenetic tree was made using the 20 RNF128 orthologs in table 1.

Ineractions

RNF128 interacts with many different proteins including CD81, TP53, USP8, USP7, TBK1, and CD151. The NSP7+NSP8 hexadecamer super complex is a SARS-Coronavirus RNA polymerase. The NSP7+NSP8 super complex is heavily involved viral replication.

[1] NCBI- https://www.ncbi.nlm.nih.gov/Structure/cdd/PF08716

Clinical Significance

-Pathlogy, disease association, mutations

In multiple studies, RNF128 is associated with p53, a tumor suppressing gene. RNF128 is seen to act negatively on P53. Multiple studies were done to show that downregulation of RNF128 lead to metastasis and a high mitotic rate in bladder and urothelial tissue.[3] Another study found that the overexpression of RNF128 can inhibit p53-induced apoptosis by degradation of p53 and thus can be linked to a regulatory mechanism for the control of p53 under stressful circumstances.[4] One other article I want mention looks at RNF128 and its role in CD4 and CD83 expression. This study shows that RNF128 is able to down regulate the expression of CD83 on CD4 T cells.[5] After searching on OMIM, I found that RNF128 expression limits IL2 and IL4 production by T lymphocytes.[6]

References

[1] Wikigenes https://www.wikigenes.org/e/gene/e/79589.html

[2] NCBI Gene https://www.ncbi.nlm.nih.gov/gene/79589

[3] Aceview- https://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/av.cgi?db=human&term=RNF128&submit=Go

[4] NCBI Gene- https://www.ncbi.nlm.nih.gov/gene/79589

[1] Lee YY, Wang CT, Huang SK, et al. Downregulation of RNF128 Predicts Progression and Poor Prognosis in Patients with Urothelial Carcinoma of the Upper Tract and Urinary Bladder. J Cancer. 2016;7(15):2187-2196. Published 2016 Oct 25. doi:10.7150/jca.16798

[2] Wei CY, Zhu MX, Yang YW, et al. Downregulation of RNF128 activates Wnt/β-catenin signaling to induce cellular EMT and stemness via CD44 and CTTN ubiquitination in melanoma. J Hematol Oncol. 2019;12(1):21. Published 2019 Mar 4. doi:10.1186/s13045-019-0711-z

[3] Lee YY, Wang CT, Huang SK, et al. Downregulation of RNF128 Predicts Progression and Poor Prognosis in Patients with Urothelial Carcinoma of the Upper Tract and Urinary Bladder. J Cancer. 2016;7(15):2187-2196. Published 2016 Oct 25. doi:10.7150/jca.16798

[4] Chen YC, Chan JY, Chiu YL, et al. Grail as a molecular determinant for the functions of the tumor suppressor p53 in tumorigenesis. Cell Death Differ. 2013;20(5):732-743. doi:10.1038/cdd.2013.1

[5] Su LL, Iwai H, Lin JT, Fathman CG. The transmembrane E3 ligase GRAIL ubiquitinates and degrades CD83 on CD4 T cells. J Immunol. 2009;183(1):438-444. doi:10.4049/jimmunol.0900204

[6] OMIM(RNF128)- https://omim.org/entry/300439

[7] http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&p=1&u=%2Fnetahtml%2FPTO%2Fsearch-bool.html&r=2&f=G&l=50&co1=AND&d=PTXT&s1=RNF128&OS=RNF128&RS=RNF128

1.Ray D, Ray P, Ferrer-Torres D, et al. Isoforms of RNF128 Regulate the Stability of Mutant P53 in Barrett's Esophageal Cells. Gastroenterology. 2020;158(3):583‐597.e1. doi:10.1053/j.gastro.2019.10.040

2. Wei CY, Zhu MX, Yang YW, et al. Downregulation of RNF128 activates Wnt/β-catenin signaling to induce cellular EMT and stemness via CD44 and CTTN ubiquitination in melanoma. J Hematol Oncol. 2019;12(1):21. Published 2019 Mar 4. doi:10.1186/s13045-019-0711-z

3. Song, G., Liu, B., Li, Z. et al. E3 ubiquitin ligase RNF128 promotes innate antiviral immunity through K63-linked ubiquitination of TBK1. Nat Immunol 17, 1342–1351 (2016). https://doi.org/10.1038/ni.3588