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. 2021 Sep 2;22(5):bbab091.
doi: 10.1093/bib/bbab091.

mi-IsoNet: systems-scale microRNA landscape reveals rampant isoform-mediated gain of target interaction diversity and signaling specificity

Li Guo  1 , Yongsheng Li  2 , Kara M Cirillo  3 , Robert A Marick  2 , Zhe Su  2 , Xing Yin  2 , Xu Hua  1 , Gordon B Mills  4   5 , Nidhi Sahni  3   6   7 , S Stephen Yi  2   8   9   10
Affiliations

mi-IsoNet: systems-scale microRNA landscape reveals rampant isoform-mediated gain of target interaction diversity and signaling specificity

Li Guo et al. Brief Bioinform. .

Abstract

MicroRNA (miRNA) is not a single sequence, but a series of multiple variants (also termed isomiRs) with sequence and expression heterogeneity. Whether and how these isoforms contribute to functional variation and complexity at the systems and network levels remain largely unknown. To explore this question systematically, we comprehensively analyzed the expression of small RNAs and their target sites to interrogate functional variations between novel isomiRs and their canonical miRNA sequences. Our analyses of the pan-cancer landscape of miRNA expression indicate that multiple isomiRs generated from the same miRNA locus often exhibit remarkable variation in their sequence, expression and function. We interrogated abundant and differentially expressed 5' isomiRs with novel seed sequences via seed shifting and identified many potential novel targets of these 5' isomiRs that would expand interaction capabilities between small RNAs and mRNAs, rewiring regulatory networks and increasing signaling circuit complexity. Further analyses revealed that some miRNA loci might generate diverse dominant isomiRs that often involved isomiRs with varied seeds and arm-switching, suggesting a selective advantage of multiple isomiRs in regulating gene expression. Finally, experimental validation indicated that isomiRs with shifted seed sequences could regulate novel target mRNAs and therefore contribute to regulatory network rewiring. Our analysis uncovers a widespread expansion of isomiR and mRNA interaction networks compared with those seen in canonical small RNA analysis; this expansion suggests global gene regulation network perturbations by alternative small RNA variants or isoforms. Taken together, the variations in isomiRs that occur during miRNA processing and maturation are likely to play a far more complex and plastic role in gene regulation than previously anticipated.

Keywords: functional diversity; gain of interactions; gene regulatory networks; isoforms; microRNA (miRNA); signaling specificity.

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Figures

Figure 1
Figure 1
Systematic characterization of deregulated isomiRs across cancer types. (A) The phenomenon of multiple isomiRs in a miRNA locus. This study mainly includes three sections to explore isomiRs: pan-cancer analysis of deregulated isomiRs, isomiR landscape profiles compared between tumor and normal samples and the most dominant isomiR expression profiles. (B) The scatter plot shows the distributions of log2FC and –log10padj values based on differential expression analysis at the isomiR level (the baseMean value in DESeq analysis of each isomiR is more than 100 in one or more cancer types). The mean value of log2FC is shown using a red dashed line. Blue dots indicate significantly downregulated isomiRs, grey dots indicate normally expressed isomiRs and red dots indicate significantly up-regulated isomiRs. The box plots (right) indicate the detailed distribution of log2FC across cancer types. The P-value is estimated using a trend test based on the median of log2FC. FC, fold change in expression; padj, adjusted P-values. (C) Distributions of the frequency of differentially expressed isomiRs in miRNA loci, length of abnormal isomiRs and number of involved seeds in miRNA loci across cancers.
Figure 2
Figure 2
IsomiR expression patterns across different cancer types. (A) Clustering analysis of differentially expressed isomiRs indicates dynamic expression (these isomiRs are abundantly expressed species, and the baseMean value of each isomiR is more than 1000 in one or more cancer types). (B) Specific examples of differentially expressed isomiR profiles in miR-10a-5p and miR-21-5p loci. For example, miR-10a-5p, 03-25 indicates the locus hg38:chr17:48579903-48579925:− and miR-21-5p, 272-294 indicates the locus hg38:chr17:59841272-59841294:+.
Figure 3
Figure 3
Functional analysis of novel seeds and their canonical seeds. (A) Some miRNA loci show significant differences between target mRNAs of novel seeds and their canonical seeds based on hypergeometric tests. (B) An example of functional analysis of isomiRs in miR-21-5p. b1: Distribution of candidate targets between total novel seeds and canonical seeds; b2: a Venn diagram of targets among detailed seeds; b3: number of CGC genes among different seeds; b4: distribution of cancer hallmarks among different seeds. (C) An example of functional analysis of isomiRs in miR-143-3p locus. c1: Distribution of targets among novel seeds and canonical seeds; c2: distribution of cancer hallmarks among different seeds.
Figure 4
Figure 4
Rewired regulatory networks for novel seeds via seed shifting. (A) The heat map shows enrichment analysis of cancer hallmarks compared between novel seeds and their canonical seeds (if both canonical and novel seeds are not significant, the miRNA locus is removed). The x-axis indicates the seven cancer hallmarks: 1, evading apoptosis; 2, insensitivity to anti-growth signals; 3, limitless replicative potential; 4, reprogramming energy metabolism; 5, self-sufficiency in growth signals; 6, sustained angiogenesis; 7, tissue invasion and metastasis. (B) Enrichment analysis of CGC and essential genes between novel seeds and their canonical seeds (if both canonical and novel seeds are not significant, the miRNA locus is removed). (C) Distribution of enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and GO terms based on total novel seeds and canonical seeds as well as distribution of enriched GO terms compared between canonical seeds and novel seeds based on each miRNA locus (screened miRNA loci are derived from Figure 3A; if both canonical and novel seeds are not significant, the miRNA locus is removed). (D) An example of a rewired regulatory network and enriched cancer hallmarks in miR-21-5p. Five seeds are contained in this network, including one canonical seed (blue diamond) and four novel seeds (red diamond; isomiR+N, indicating that the canonical seed is shifted to 3′ ends by N nt). This network is constructed on the basis of seeds; indeed, every seed may involve multiple 3′ isomiR sequences. The most dominant enriched cancer hallmark is presented for specific targets.
Figure 5
Figure 5
Dominant isomiR sequence analysis and arm-switching phenomenon based on pre-miRNA. (A) Pie charts indicate change in number of isomiRs across different cancer types. ‘Common’ indicates number of dominant isomiRs shared between tumor and normal samples, ‘normal’ indicates number of dominant isomiRs in normal samples, ‘tumor’ indicates number of dominant isomiRs in tumor samples and ‘different’ indicates number of dominant isomiRs differing between tumor and normal samples. (B) Frequency distributions of the most dominant isomiRs from miR-30e-5p and miR-30e-3p (based on frequencies of samples). These involved isomiRs were found to be the most dominant isomiRs in specific samples, and the detailed distribution across cancer types is also presented. 71-90 indicates a location of hg38:chr1:40754371-40754390:+, and 13-33 indicates hg38:chr1:40754413-40754433:+. (C) Distribution of abnormally expressed isomiRs from two arms of mir-30e. Scatter plot indicates expression variation between miR-30e-5p and miR-30e-3p across cancer types based on expression levels of the most dominant isomiRs generated from miR-30e-5p and miR-30e-3p.
Figure 6
Figure 6
Survival analysis shows divergence among different isomiRs. (A) An example of survival analysis using different isomiRs in miR-21-5p and their expression patterns. Survival results vary between different isomiRs. 73-94 indicates a location of hg38:chr17:59841273-59841294:+. (B) Another example of survival analysis using different isomiRs in miR-30e-5p and their expression patterns. Survival results again vary between different isomiRs. In LAML, no normal samples are involved; ‘All samples’ represent samples involved from 33 cancer types. 71-90 indicates a location of hg38:chr1:40754371-40754390:+.
Figure 7
Figure 7
Experimental validation of biological function of isomiRs with novel seeds. (A) miR-101-3p locus is selected to perform further experimental validation. Paired analysis is performed on the canonical miRNA and its isomiR with a novel seed to show their specific expression patterns across diverse cancer types. (B) RAB11FIP1 mRNA levels are assessed after treatment with canonical miRNA or its isomiR mimics with RT-qPCR. nc, negative control. (C) Protein levels of RAB11FIP1 are evaluated after treatment with canonical miRNA or its isomiR mimics with western blotting.
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