Relevance of 2′-O-Methylation and Pseudouridylation for the Malignant Melanoma

Simple Summary This study investigates the expression, the histological localization, and the influence of the factors involved in 2′-O-methylation and pseudouridylation on prognostic relevant markers, proliferation markers, overall survival, molecular immune surveillance and evasion mechanisms within the malignant melanoma. Statistically significant positive correlations to the expression of markers involved in cell proliferation were observed. The upregulation of the RNA modifying factors was of prognostic relevance in this tumor disease with a negative impact on the overall survival of melanoma patients. Furthermore, the factors involved in 2′-O-methylation and pseudouridylation were statistically significant negative correlated to the expression of human leukocyte antigen class I genes as well as of components of the antigen processing machinery. Abstract The two RNA modifications 2′-O-methylation and pseudouridylation occur on several RNA species including ribosomal RNAs leading to an increased translation as well as cell proliferation associated with distinct functions. Using malignant melanoma (MM) as a model system the proteins mediating these RNA modifications were for the first time analyzed by different bioinformatics tools and public available databases regarding their expression and histological localization. Next to this, the impact of these RNA-modifying factors on prognostic relevant processes and marker genes of malignant melanoma was investigated and correlated to immune surveillance and evasion strategies. The RNA modifying factors exerted statistically significant positive correlations to the expression of genes involved in cell proliferation and were statistically significant negative correlated to the expression of human leukocyte antigen class I genes as well as of components of the antigen processing machinery in malignant melanoma. Upregulation of the RNA modifying proteins was of prognostic relevance in this tumor disease with a negative impact on the overall survival of melanoma patients. Furthermore, the expression of known oncogenic miRs, which are induced in malignant melanoma, directly correlated to the expression of factors involved in these two RNA modifications.


Introduction
Malignant melanoma (MM) refers to a neoplasm of melanocytes comprising of neural crest-derived cells located in particular in the stratum basal of the skin's epidermis and in the uvea of the eye. In contrast to keratinocytes, melanocytes are not linked to the basal lamina by hemidesmosomes and do not have desmosomes to neighboring keratinocytes. Instead, melanocytes as well as adjacent keratinocytes express, e.g., E-cadherin on their cell surface [1], which is an important factor contributing to the cell migration and invasion of melanoma cells after malignant transformation of melanocytes. Due to this migratory ability, growth characteristics as well as other properties including resistance to radiation dine synthase is PUS10 involved in the miR maturation, which results after its depletion in a reduced expression of mature miRs [31]. Furthermore, the 2′-O-methylation and the pseudouridylation have also an impact on the binding of RNA-binding proteins (RBPs) and are required for an appropriate mRNA splicing [25,30], which is altered in various tumor entities including MM [32][33][34]. In this study, the expression of key molecules involved in 2′-O-methylation and pseudouridylation is addressed in more detail using MM as a model.  Many sites of the pseudouridylation are evolutionarily conserved [26,27]. In humans, the uridine is converted to pseudouridine in rRNA, sno/scaRNA and snRNA by dyskerin (DKC1), which is a component of a complex consisting of one H/ACA snoRNA and four core proteins, namely GAR1 ribonucleoprotein (GAR1), NHP2 ribonucleoprotein (NHP2), NOP10 ribonucleoprotein (NOP10) and DKC1 itself [28]. The substitution of uridine with pseudouridine introduces a novel H bond donor on the non-Watson Crick site of the nucleotide affecting the secondary structure and consequently structure related interactions [29] ( Figure 1B). Pseudouridylation is involved in many important processes of gene expression including spliceosomal small nuclear ribonucleoprotein (snRNP) biogenesis, efficiency of pre-mRNA splicing and translation fidelity [30]. Another human pseudouridine synthase is PUS10 involved in the miR maturation, which results after its depletion in a reduced expression of mature miRs [31]. Furthermore, the 2 -O-methylation and the pseudouridylation have also an impact on the binding of RNA-binding proteins (RBPs) and are required for an appropriate mRNA splicing [25,30], which is altered in various tumor entities including MM [32][33][34]. In this study, the expression of key molecules involved in 2 -O-methylation and pseudouridylation is addressed in more detail using MM as a model.

Correlation of RNA-Modifying Proteins with Tumor Cell Proliferation
To address whether the expression of factors involved in 2′-O-methylation and pseudouridylation correlates with the pathological increased proliferation rates in MM, the expression data of these RNA-modifying factors were analyzed in The Cancer Genome Atlas (TCGA) data sets consisting of 214 samples from MM patients provided by the R2 data base. The following proliferation markers known to play a role in MM were investi-

Correlation of RNA-Modifying Proteins with Tumor Cell Proliferation
To address whether the expression of factors involved in 2 -O-methylation and pseudouridylation correlates with the pathological increased proliferation rates in MM, the expression data of these RNA-modifying factors were analyzed in The Cancer Genome Atlas (TCGA) data sets consisting of 214 samples from MM patients provided by the R2 data base. The following proliferation markers known to play a role in MM were investigated: Ki67 (MKI67), PCNA, cyclin A (CCNA1), cyclin B (CCNB1), MCM2, MCM4, and mitosin (CENPF) [35][36][37][38].
As summarized in Table 2, positive correlations between both RNA modifying factors and the proliferation markers analyzed were found. The effect was the strongest for MCM4, which correlated statistically significant to all factors involved into 2 -O-methylation and pseudouridylation with the exception of NOP10. In addition, PCNA, cyclin B and mitosin showed strong correlations, while the expression of CCNA1 did not correlate statistically significant with any of these factors.
Based on the strong positive correlation between the factors involved in 2 -O-methylation and pseudouridylation with most of the clinical relevant proliferation markers, a possible correlation between these factors and prognostic marker genes relevant for MM was evaluated such as melanoma antigen recognized by T cells (MART)1, S100 calcium binding protein B (S100B), S100A4, S100A9, melanocyte inducing transcription factor (MITF), matrix metallopeptidase 2 (MMP2), nucleoside diphosphate kinase 1 (NM23), cluster of differentiation (CD) 44, premelanosome protein (PMEL), and BCL2 apoptosis regulator (BCL2) using the same TCGA data sets [39][40][41][42][43] (Table 3). Next to these marker genes the invasion depth termed Breslow's depth is of prognostic value. The average Breslow score of the 214 MM patients analyzed was 2.5 mm. All other clinical parameters of this MM patient cohort (n = 214) of the analyzed microarray data can be obtained from the original literature Jönsson et al., 2015 [44].
The two markers MART1 and MITF, but also NM23 showed statistically significant positive correlations to the expression of RNA modifying proteins. In contrast, the S100 family members S100B, S100A4, S100A9 and MMP2 exhibiting a prognostic potential in MM exerted a statistically significant negative correlation, whereas CD44 did not show any correlation and PMEL as well as BCL2 were only weakly positivey correlated.
To address whether the factors involved in 2 -O-methylation and pseudouridylation are of central importance for prognosis in MM, the same TCGA data set was used for the generation of Kaplan-Meier plots. As shown in Figure 4A-H, a direct correlation of the expression level of these factors with the overall survival (OS) of MM patients was detected and statistically significant for FBL, NOP58, and GAR1. In contrast, low NOP10 expression levels predicted a statistically non-significant correlation with a worse patients' outcome ( Figure 4). However, it is noteworthy that the use of TCGA data cannot replace extensive protein based analyses of ex vivo MM specimens.

Correlation of the Expression of RNA Modifying Factors with Immune Modulatory Genes
Due to the increased implementation of immunotherapies for the treatment of hematopoietic and solid tumors, it was analyzed whether the factors involved in 2 -O-methylation and pseudouridylation directly or indirectly affect transcripts participating in the immune surveillance of tumor cells. Therefore, their expression patterns were correlated to molecules involved in the immune recognition/evasion of malignant and/or virus transformed cells from immune effector cells. Using the same melanoma data set, an impressive statistically significant negative correlation between immunological relevant molecules, in particular the human leucocyte antigen (HLA) class Ia and Ib and components of the HLA class I antigen processing machinery (APM), with the factors involved in 2 -O-methylation and pseudouridylation was detected (Table 4). This might also explain the negative correlation of these factors with the OS of MM patients, since their impaired expression was associated with a reduced anti-tumoral immune cell response [45] (Figure 4). The divergent statistically significant positive correlation pattern of NOP10 to molecules involved in immune surveillance was also associated with a statistically significant positive correlation to the OS in MM patients ( Figure 4H). How-ever, further studies are needed to investigate whether the statistically significant negative correlated immunological relevant genes are directly negatively regulated upon a reduced activity/expression of the factors involved 2 -O-methylation and pseudouridylation.
MM exerted a statistically significant negative correlation, whereas CD44 did not show any correlation and PMEL as well as BCL2 were only weakly positivey correlated.
To address whether the factors involved in 2′-O-methylation and pseudouridylation are of central importance for prognosis in MM, the same TCGA data set was used for the generation of Kaplan-Meier plots. As shown in Figure 4A-H, a direct correlation of the expression level of these factors with the overall survival (OS) of MM patients was detected and statistically significant for FBL, NOP58, and GAR1. In contrast, low NOP10 expression levels predicted a statistically non-significant correlation with a worse patients' outcome ( Figure 4). However, it is noteworthy that the use of TCGA data cannot replace extensive protein based analyses of ex vivo MM specimens.

Correlation of the Expression of RNA Modifying Factors with Immune Modulatory Genes
Due to the increased implementation of immunotherapies for the treatment of hematopoietic and solid tumors, it was analyzed whether the factors involved in 2′-O-methylation and pseudouridylation directly or indirectly affect transcripts participating in the immune surveillance of tumor cells. Therefore, their expression patterns were correlated to molecules involved in the immune recognition/evasion of malignant and/or virus transformed cells from immune effector cells. Using the same melanoma data set, an impressive statistically significant negative correlation between immunological relevant molecules, in particular the human leucocyte antigen (HLA) class Ia and Ib and components of the HLA class I antigen processing machinery (APM), with the factors involved in 2′-Omethylation and pseudouridylation was detected (Table 4). This might also explain the negative correlation of these factors with the OS of MM patients, since their impaired expression was associated with a reduced anti-tumoral immune cell response [45] ( Figure  4). The divergent statistically significant positive correlation pattern of NOP10 to molecules involved in immune surveillance was also associated with a statistically significant positive correlation to the OS in MM patients ( Figure 4H). However, further studies are

Correlation of miR Expression with RNA-Modifying Factors
The processes of 2 -O-methylation and pseudouridylation also occur in other RNA species with a strong impact to diverse molecular processes of malignant transformation and thus are of clinical relevance. These other RNA species include microRNAs (miRs), which are non-coding single stranded RNAs with an approximately length of~22 nt [46], binding specifically and preferentially, but not exclusively, within the 3 -untranslated region (UTR) to their target mRNAs sequence [40,47] thereby causing a translational repression and mRNA decay [48].
Some miRs can be classified into oncogenic, tumor suppressive and/or immune modulatory miRs [48]. In addition, human viruses including herpes viruses encode for viral miRs, which also affect cancer related cellular processes [49].
Thus far, only little is known, which of these enzymes are able to modify miRs and which alterations in the respective miR-mediated functions and/or miR stability are caused by such modifications.
To determine whether the expression of known oncogenic miRs in MM might be correlated with the expression of enzymes involved into 2 -O-methylation and pseudouridylation, the TCGA data of 214 human MM samples were further analyzed for miR expression. As shown in Table 5, there existed no evidence for a global impact of the induction of oncogenic miRs, while miR-21 was statistically significant negatively correlated to the expression of the investigated enzymes in MM. Table 5. Correlation of miR expression with enzymes known to be involved in 2 -O-methylation or pseudouridylation of miRs. Analysis of the TCGA data from 214 human melanoma tumors provided by the R2 database (https://hgserver1.amc.nl/ (accessed on 12 February 2021)) for correlation with enzymes known to be involved in miR 2 -O-methylation and pseudouridylation with the amount of increased and/or stabilized oncogenic miRs reported to be induced in MM.

Discussion
Recently, the aberrant expression pattern of DKC1, NHP2, and NOP10 in several cancer entities has been reviewed [52]. Due to their biological functions, the localization of these factors was almost completely limited to the nucleus, since 2 -O-methylation and pseudouridylation occur within the nucleoli. Furthermore, an increase within the highly proliferational active cells of the epidermal stratum germinativum was demonstrated, which reflects the involvement and increased number of ribosomes during proliferation. This was further underlined by statistically significant positive correlations to proliferation markers suggesting that some factors involved in 2 -O-methylation and pseudouridylation might also be suitable markers for proliferation themselves.
However, this study did not answer whether in the case of positive correlations between the factors involved in 2 -O-methylation and pseudouridylation and, e.g., proliferation markers these positive correlations are an indirect result due to an enhanced proliferation per se mediated by increased translational efficacy caused by modifications within ribosomal RNAs, or whether the mRNAs of the positively correlated proliferation markers are directly modified by these factors or by both mechanisms in parallel.
The positive correlations described in this study are based upon TCGA data, not taken posttranscriptional mechanisms of gene regulation into account. Therefore, in depth protein-based studies applying human melanoma tissue specimens are required to proof this hypothesis.
In regard to the observed statistically significant negative and coordinated correlations to molecules involved in immune surveillance/evasion it is noteworthy that (i) HLA Ia and Ib genes, HLA class related MICs and major APM components are located within the major histocompatibility complex (MHC) locus on chromosome 6p21 and that (ii) the adaptive immunity phylogenetically occurs with the jawed vertebrates [53] and, e.g., HLA class Ib genes are evolutionary even younger, which might have an impact on the observed negative correlations.
Due to the limitation of transcriptome based microarray data sets it is necessary to underline that also indirect effects may cause such down regulations. The increased proliferation itself might decrease the antigen processing and presentation efficacy in melanoma cells. In turn, the impaired antigen processing and presentation is a frequent immune evasion strategy of tumors [54], which might be a prerequisite for an increased proliferation rate of tumor cells. Thus far, the sequence of these events in MM has not been studied.
RNA modifications on miRs affecting their stability or decay might also indirectly contribute to the regulation of cancer related processes, even independently of the translational processes linked to the number of ribosomes. This opens a new regulatory dimension for RNA modifying enzymes ( Figure 5), which has to be explored in more detail.
Thus far, it is controversially discussed whether mature mammalian miRs contain 2 -O-methylations as reported for plants and Drosophila [55]. In Drosophila, 2 -O-methylation of miRs occurred age dependent and its inhibition resulted in an accelerated neurodegeneration and shorter life span [56]. Despite discrepancies regarding the missing 2 -Omethylation in mammalian miRs, the human miR-21-5p has been shown to contain a 3 -terminal 2 -O-methylation, which enhances the stability of this oncogenic miR in lung cancer patients. Interestingly, HENMT1 was identified to act as methyltransferase [57].
Indirect effects concerning the positive or negative correlations of factors involved in 2 -O-methylation and pseudouridylation with miRs might occur. Several miRs are processed from introns after the splicing, while for the process of splicing the snoRNAs as huge group of the snRNAs in involved. These snoRNAs harbor themselves 2 -O-methylations and pseudouridylations contributing to the stability and functionality of the snoRNAs.
In this study, the impact and relevance of factors involved in 2 -O-methylation and pseudouridylation of different RNA species related to the processes of tumor formation and progression were summarized in MM as a tumor model. Using different bioinformatics tools statistically significant positive correlations between proliferation and prognostic marker genes relevant for the MM with the factors involved into 2 -O-methylation and pseudouridylation were described for the first time.
The impact of these molecules on other RNA species including miRs has recently been investigated, but needs to be further studied in detail. This will lead to the identification of proteins involved in such miR modifications, which might have an impact on the function of such modified miRs. However, the existence of miR 2 -O-methylation and pseudouridylation in general must not necessarily involve tumor relevant miRs and processes.
class Ib genes are evolutionary even younger, which might have an impact on the observed negative correlations.
Due to the limitation of transcriptome based microarray data sets it is necessary to underline that also indirect effects may cause such down regulations. The increased proliferation itself might decrease the antigen processing and presentation efficacy in melanoma cells. In turn, the impaired antigen processing and presentation is a frequent immune evasion strategy of tumors [54], which might be a prerequisite for an increased proliferation rate of tumor cells. Thus far, the sequence of these events in MM has not been studied.
RNA modifications on miRs affecting their stability or decay might also indirectly contribute to the regulation of cancer related processes, even independently of the translational processes linked to the number of ribosomes. This opens a new regulatory dimension for RNA modifying enzymes ( Figure 5), which has to be explored in more detail. Thus far, it is controversially discussed whether mature mammalian miRs contain 2′-O-methylations as reported for plants and Drosophila [55]. In Drosophila, 2′-O-methylation

Materials and Methods Immunohistochemistry
The frequency and localization of protein levels involved in the two RNA modification processes, namely FBL, NOP56, NOP58, 15.5K (SNU13), DKC1, GAR1, NHP2, and NOP10, were analyzed by evaluation of immunohistochemistry data of healthy normal skin sections and melanoma specimen provided by the free available online data base. The Human Protein Atlas [58][59][60].
Bioinformatic analyses of gene expression data and correlation with patients' overall survival.
The factors involved in RNA 2 -O-methylation and pseudouridylation were correlated with genes encoding for above mentioned ribosomal RNAs carrying multiple of such modifications. Unfortunately, the applied Microarrays (Illumina Human HT-12V4.0 Chips) of the 214 MM patients published by Jönsson and co-authors 2015 [44] contained only probes against the human RN5S9 transcript encoding the 5S ribosomal RNA. The factors showed a positive correlation to the RN5S9, which was statistically significant for NOP56, NOP58 and NHP2.
The correlation coefficients reflecting R values and the respective p values were calculated by the R2 database (https://hgserver1.amc.nl/ (accessed on 12 February 2021)). For visualization these R values were presented in a heatmap generated by GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA).
Correlation of the expression pattern of genes involved into 2 -O-methylation and pseudouridylation with preselected genes involved in cell proliferation, prognosis and immune recognition/evasion in MM is based upon TCGA data of 214 melanoma specimens provided by the R2 database (https://hgserver1.amc.nl/ (accessed on 12 February 2021)). Statistically significant positive or negative correlations (p < 0.05) are highlighted in red or green. The same TCGA data set was applied for the generation of the respective Kaplan-Meier Plots.

Conclusions
Thus, a link between 2 -O-methylation and pseudouridylation to cell proliferation, host immunity and oncogenic miRs exists in MM suggesting that both RNA modifications and factors involved in this process represent suitable targets for tumor therapy and putative novel prognostic markers.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.