Two for one: regulatory RNAs that encode small proteins.

RNAs are commonly categorized as being either protein-coding mRNAs or noncoding RNAs. However, an increasing number of transcripts, in organisms ranging from bacteria to humans, are being found to have both coding and noncoding functions. In some cases, the sequences encoding the protein and the regulatory RNA functions are separated, while in other cases the sequences overlap. The protein and RNA can regulate similar or distinct pathways. Here we describe examples illustrating how these dual-function (also denoted bifunctional or dual-component) RNAs are identified and their mechanisms of action and cellular roles. We also discuss the synergy or competition between coding and RNA activity and how these regulators evolved, as well as how more dual-function RNAs might be discovered and exploited.

Multi-omic Mitoprotease Profiling Defines a Role for Oct1p in Coenzyme Q Production.

Mitoproteases are becoming recognized as key regulators of diverse mitochondrial functions, although their direct substrates are often difficult to discern. Through multi-omic profiling of diverse Saccharomyces cerevisiae mitoprotease deletion strains, we predicted numerous associations between mitoproteases and distinct mitochondrial processes. These include a strong association between the mitochondrial matrix octapeptidase Oct1p and coenzyme Q (CoQ) biosynthesis-a pathway essential for mitochondrial respiration. Through Edman sequencing and in vitro and in vivo biochemistry, we demonstrated that Oct1p directly processes the N terminus of the CoQ-related methyltransferase, Coq5p, which markedly improves its stability. A single mutation to the Oct1p recognition motif in Coq5p disrupted its processing in vivo, leading to CoQ deficiency and respiratory incompetence. This work defines the Oct1p processing of Coq5p as an essential post-translational event for proper CoQ production. Additionally, our data visualization tool enables efficient exploration of mitoprotease profiles that can serve as the basis for future mechanistic investigations.

Light-dependent expression and accumulation of miR408-encoded peptide, miPEP408, is regulated by HY5 in Arabidopsis.

Recent studies propose that primary transcripts of miRNAs (pri-miRNAs) contain small Open Reading Frames (ORFs) capable of encoding miRNA-encoded peptides (miPEPs). These miPEPs can function as transcriptional regulators for their corresponding pri-miRNAs, ultimately enhancing mature miRNA accumulation. Notably, pri-miR408 encodes the functional peptide miPEP408, regulating expression of miR408 and its target genes, providing plant tolerance to stresses. While miPEPs are crucial regulators, the factors governing them are have not been studied in detail. Here, we explored the light-dependent regulation of miPEP408 in Arabidopsis. Expression analysis during dark-light transitions revealed light-induced transcription and accumulation of the miPEP408. As the promoter of miR408 contains cis-acting elements responsible for binding to the bZIP-type transcription factor ELONGATED HYPOCOTYL5 (HY5), known for light-mediated regulation in plants, we studied its involvement in the regulation of miR408. Analysis of HY5 mutant (hy5-215), complemented line (HY5OX/hy5), and CONSTITUTIVE PHOTOMORPHOGENIC 1 mutant (cop1-4) plants supported HY5's positive regulation of miPEP408. Grafting and GUS assays further suggested the role of HY5 as a shoot-root mobile signal inducing light-dependent miPEP408 expression. This study underscores the regulatory impact of light on small peptides, exemplified by miPEP408, mediated by the key transcription factor HY5.

MicroRNA-encoded regulatory peptides modulate cadmium tolerance and accumulation in rice.

MicroRNAs (miRNAs) are small noncoding RNAs that play a vital role in plant responses to abiotic and biotic stresses. Recently, it has been discovered that some primary miRNAs (pri-miRNAs) encode regulatory short peptides called miPEPs. However, the presence of miPEPs in rice, and their functions in response to abiotic stresses, particularly stress induced by heavy metals, remain poorly understood. Here, we identified a functional small peptide (miPEP156e) encoded by pri-miR156e that regulates the expression of miR156 and its target SPL genes, thereby affecting miR156-mediated cadmium (Cd) tolerance in rice. Overexpression of miPEP156e led to decreased uptake and accumulation of Cd and reactive oxygen species (ROS) levels in plants under Cd stress, resulting in improved rice Cd tolerance, as observed in miR156-overexpressing lines. Conversely, miPEP156e mutants displayed sensitivity to Cd stress due to the elevated accumulation of Cd and ROS. Transcriptome analysis further revealed that miPEP156e improved rice Cd tolerance by modulating Cd transporter genes and ROS scavenging genes. Our study provides insights into the regulatory mechanism of miPEP156e in rice response to Cd stress and demonstrates the potential of miPEPs as an effective tool for improving crop abiotic stress tolerance.

ESF1 and MIPEP proteins promote estrogen receptor-positive breast cancer proliferation and are associated with patient prognosis.

BACKGROUND: Estrogen receptor-positive (ER+) breast cancer accounts for two-thirds of all breast cancers, and its early and late recurrences still threaten patients' long-term survival and quality of life. Finding candidate tumor antigens and potential therapeutic targets is critical to addressing these unmet needs. METHOD: The isobaric tags for relative and absolute quantitation (iTRAQ) proteomic analysis was employed to identify the differentially expressed proteins (DEPs) between ER + breast cancer and corresponding adjacent normal tissue. Candidate DEPs were screened by bioinformatic analyses, and their expression was confirmed by immunohistochemical (IHC) staining and western blot. A series of in vitro experiments, including wound healing assay, colony formation, and cell cycle assay, were performed to reveal the functions of selected DEPs. Additionally, their clinical significances were further analyzed. RESULT: A total of 369 DEPs (fold change ≥ 2.0 or ≤ 0.66, P < 0.05) were discovered. Compared with normal tissue, 358 proteins were up-regulated and 11 proteins were down-regulated in ER + breast cancer. GO and KEGG enrichment analysis showed that DEPs were closely associated with RNA regulation and metabolic pathways. STRING analysis found ESF1 and MIPEP were the hub genes in breast cancer, whose increased expressions were verified by the IHC staining and western blot. Knocking down ESF1 and MIPEP inhibited colony formation and increased cell apoptosis. Besides, knocking down ESF1 inhibited wound healing but not MIPEP. In addition, ESF1 and MIPEP expression were negatively associated with patient prognosis. CONCLUSION: The upregulation of ESF1 and MIPEP promoted ER + breast cancer proliferation, which might provide novel targets for the development of new therapies.

miPEP31 alleviates Ang II-induced hypertension in mice by occupying Cebpα binding sites in the pri-miR-31 promoter.

BACKGROUND: Previous studies have shown that peptides encoded by noncoding RNAs (ncRNAs) can be used as peptide drugs to alleviate diseases. We found that microRNA-31 (miR-31) is involved in the regulation of hypertension and that the peptide miPEP31, which is encoded by the primary transcript of miR-31 (pri-miR-31), can inhibit miR-31 expression. However, the role and mechanism of miPEP31 in hypertension have not been elucidated. METHODS: miPEP31 expression was determined by western blot analysis. miPEP31-deficient mice (miPEP31-/-) were used, and synthetic miPEP31 was injected into Ang II-induced hypertensive mice. Blood pressure was monitored through the tail-cuff method. Histological staining was used to evaluate renal damage. Regulatory T (Treg) cells were assessed by flow cytometry. Differentially expressed genes were analysed through RNA sequencing. The transcription factors were predicted by JASPAR. Luciferase reporter and electrophoretic mobility shift assays (EMSAs) were used to determine the effect of pri-miR-31 on the promoter activity of miPEP31. Images were taken to track the entry of miPEP31 into the cell. RESULTS: miPEP31 is endogenously expressed in target organs and cells related to hypertension. miPEP31 deficiency exacerbated but exogenous miPEP31 administration mitigated the Ang II-induced systolic blood pressure (SBP) elevation, renal impairment and Treg cell decreases in the kidney. Moreover, miPEP31 deletion increased the expression of genes related to Ang II-induced renal fibrosis. miPEP31 inhibited the transcription of miR-31 and promoted Treg differentiation by occupying the Cebpα binding site. The minimal functional domain of miPEP31 was identified and shown to regulate miR-31. CONCLUSION: miPEP31 was identified as a potential therapeutic peptide for treating hypertension by promoting Treg cell differentiation in vivo. Mechanistically, we found that miPEP31 acted as a transcriptional repressor to specifically inhibit miR-31 transcription by competitively occupying the Cebpα binding site in the pri-miR-31 promoter. Our study highlights the significant therapeutic effect of miPEP31 on hypertension and provides novel insight into the role and mechanism of miPEPs.

Gene silencing in broomrapes and other parasitic plants of the Orobanchaceae family: mechanisms, considerations, and future directions.

Holoparasites of the Orobanchaceae family are devastating pests causing severe damage to many crop species and are nearly impossible to control with conventional methods. During past decades RNA interference (RNAi) has been seen as a promising approach to control various crop pests. The exchange of small RNAs (sRNAs) between crops and parasitic plants has been documented indicating a potential for the development of methods to protect them via the delivery of the sRNAs to parasites, called host-induced gene silencing (HIGS). Here we describe various approaches used for gene silencing in plants and suggest solutions to improve the long-distance movement of the silencing triggers to elevate the HIGS efficiency in parasitic plants. We also investigate the important biological processes during parasites life cycle with a focus on broomrape species, providing several appropriate target genes that can be used in, especially, multiplex gene silencing experiments. We also touch on how the application of nanoparticles can improve the stability and delivery of the silencing triggers, highlighting its potential for parasitic plants control. Finally, suggestions for further research and possible directions for RNAi in parasitic plants are provided.

A peptide encoded by pri-miRNA-31 represses autoimmunity by promoting Treg differentiation.

Recent evidence has revealed that small polypeptides (containing fewer than 100 amino acids) can be translated from noncoding RNAs (ncRNAs), which are usually defined as RNA molecules that do not encode proteins. However, studies on functional products translated from primary transcripts of microRNA (pri-miRNA) are quite limited. Here, we describe a peptide termed miPEP31 that is encoded by pri-miRNA-31. miPEP31 is highly expressed in Foxp3+ regulatory T cells (Tregs ) and significantly promotes the differentiation of Tregs without affecting their inhibitory ability. Our results show that miPEP31 is a cell-penetrating peptide both in vitro and in vivo. miPEP31 downregulates miR-31 expression, enhances peripheral Treg induction, and dramatically suppresses experimental autoimmune encephalomyelitis. Mechanistically, we show that miPEP31 acts as a transcriptional repressor inhibiting the expression of miRNA-31, a negative regulator of Tregs . Our results reveal an indispensable role of miPEP31 in maintaining immune homeostasis by promoting Treg differentiation and also present a potential therapeutic peptide for modulating miRNA expression and treating autoimmune diseases.

Regulatory miPEP Open Reading Frames Contained in the Primary Transcripts of microRNAs.

This review aims to consider retrospectively the available data on the coding properties of pri-microRNAs and the regulatory functions of their open reading frames (ORFs) and the encoded peptides (miPEPs). Studies identifying miPEPs and analyzing the fine molecular mechanisms of their functional activities are reviewed together with a brief description of the methods to identify pri-miRNA ORFs and the encoded protein products. Generally, miPEPs have been identified in many plant species of several families and in a few animal species. Importantly, molecular mechanisms of the miPEP action are often quite different between flowering plants and metazoan species. Requirement for the additional studies in these directions is highlighted by alternative findings concerning negative or positive regulation of pri-miRNA/miRNA expression by miPEPs in plants and animals. Additionally, the question of how miPEPs are distributed in non-flowering plant taxa is very important for understanding the evolutionary origin of such micropeptides. Evidently, further extensive studies are needed to explore the functions of miPEPs and the corresponding ORFs and to understand the full set of their roles in eukaryotic organisms. Thus, we address the most recent integrative views of different genomic, physiological, and molecular aspects concerning the expression of miPEPs and their possible fine functions.

MicroRNA: noncoding but still coding, another example of self-catalysis.

MicroRNAs (miRNAs) are known to interact with specific mRNAs to regulate gene expression at the post-transcriptional level by cleaving/repressing the translation process. MiRNA-mediated regulation of gene expression has become an interesting area of research on biological processes like growth, development, and stress responses. Studies suggest that some of the noncoding RNAs possess short open reading frames (ORFs) that code for micropeptides (miPEPs) having a regulatory function. Dual functions of some MIR genes are being deciphered, wherein the gene is transcribed into a longer transcript having a stem-loop structure and a shorter alternatively spliced transcript with no stem-loop. While the longer transcript is processed into miRNA, the shorter one is translated into miPEP. The miPEP enhances the transcription/production of the pri-miRNA from which it originates. Regulatory action of miPEP being species-specific, synthetic miPEP being is tested for exogenous application on crop plant to improve stress tolerance/agronomic performance. Deployment of the miPEP-mediated regulatory function might be a promising strategy to modulated miRNA-facilitated regulation of gene/trait of interest towards developing climate-resilient crops. In this review, we describe the newly identified and verified function of the MIR gene in the coding of miPEPs along with the comparison of the features of miRNA and miPEP in plant. We also discuss about their potential role in crop improvement and some of the yet unanswered question about miPEP.

HY5 regulates light-dependent expression and accumulation of miR858a-encoded peptide, miPEP858a.

microRNA encoded peptide (miPEP) has been shown to have potential to regulate corresponding miRNA and associated function. miPEP858a regulate phenylpropanoid pathway and plant development. Several studies have suggested that various factors like light, temperature, heavy metals etc. can regulate gene and their associated functions. However, what are the regulators of miPEP are not reported till date. In this study we have reported that light directly regulates miPEP858a accumulation in Arabidopsis thaliana. Peptide assay in light and dark clearly showed the essential requirement of light. Along with this, we have reported that HY5 a shoot-to-root mobile, light-mediated transcription factor plays a crucial role in the function of miPEP858a. The transcript and endogenous protein accumulation of miPEP858a in hy5-215, OXHY5/hy5, and cop1-4 suggested that the HY5 positively regulates miPEP858a. In addition to that this study also include grafting assay between shoot of different mutant and transgenic lines with root of miPEP858a promoter:reporter lines and promoter deletion construct experiment clearly suggested that HY5 a transcription factor regulates light-dependent expression and accumulation of miPEP858a.

Variants in the MIPEP gene presenting with complex neurological phenotype without cardiomyopathy, impair OXPHOS protein maturation and lead to a reduced OXPHOS abundance in patient cells.

Most mitochondrial proteins are synthesized in the cytosol and targeted to mitochondria via N-terminal mitochondrial targeting signals (MTS) that are proteolytically removed upon import. Sometimes, MTS removal is followed by a cleavage of an octapeptide by the mitochondrial intermediate peptidase (MIP), encoded by the MIPEP gene. Previously, MIPEP variants were linked to four cases of multisystemic disorder presenting with cardiomyopathy, developmental delay, hypotonia and infantile lethality. We report here a patient carrying compound heterozygous MIPEP variants-one was not previously linked to mitochondrial disease-who did not have cardiomyopathy and who is alive at the age of 20 years. This patient had developmental delay, global hypotonia, mild optic neuropathy and mild ataxia. Functional characterization of patient fibroblasts and HEK293FT cells carrying MIPEP hypomorphic alleles demonstrated that deficient MIP activity was linked to impaired post-import processing of subunits from four of the five OXPHOS complexes and decreased abundance and activity of some of these complexes in human cells possibly underlying the development of mitochondrial disease. Thus, our work expands the genetic and clinical spectrum of MIPEP-linked disease and establishes MIP as an important regulator of OXPHOS biogenesis and function in human cells.

Evidence That Regulation of Pri-miRNA/miRNA Expression Is Not a General Rule of miPEPs Function in Humans.

Some miRNAs are located in RNA precursors (pri-miRNAs) annotated as long non-coding (lncRNAs) due to absence of long open reading frames (ORFs). However, recent studies have shown that some lnc pri-miRNAs encode peptides called miPEPs (miRNA-encoded peptides). Initially discovered in plants, three miPEPs have also been identified in humans. Herein, we found that a dozen human pri-miRNAs potentially encode miPEPs, as revealed by ribosome profiling and proteomic databases survey. So far, the only known function of plant miPEPs is to enhance the transcription of their own pri-miRNAs, thereby increasing the level and activity of their associated miRNAs and downregulating the expression of their target genes. To date, in humans, only miPEP133 was shown to promote a positive autoregulatory loop. We investigated whether other human miPEPs are also involved in regulating the expression of their miRNAs by studying miPEP155, encoded by the lnc MIR155HG, miPEP497, a sORF-encoded peptide within lnc MIR497HG, and miPEP200a, encoded by the pri-miRNA of miR-200a/miR-200b. We show that overexpression of these miPEPs is unable to impact the expression/activity of their own pri-miRNA/miRNAs in humans, indicating that the positive feedback regulation observed with plant miPEPs and human miPEP133 is not a general rule of human miPEP function.

Identification of miPEP133 as a novel tumor-suppressor microprotein encoded by miR-34a pri-miRNA.

BACKGROUND: Very few proteins encoded by the presumed non-coding RNA transcripts have been identified. Their cellular functions remain largely unknown. This study identifies the tumor-suppressor function of a novel microprotein encoded by the precursor of miR-34a. It consists of 133 amino acid residues, thereby named as miPEP133 (pri-microRNA encoded peptide 133). METHODS: We overexpressed miPEP133 in nasopharyngeal carcinoma (NPC), ovarian cancer and cervical cancer cell lines to determine its effects on cell growth, apoptosis, migration, or invasion. Its impact on tumor growth was evaluated in a xenograft NPC model. Its prognostic value was analyzed using NPC clinical samples. We also conducted western blot, immunoprecipitation, mass spectrometry, confocal microscopy and flow cytometry to determine the underlying mechanisms of miPEP133 function and regulation. RESULTS: miPEP133 was expressed in normal human colon, stomach, ovary, uterus and pharynx. It was downregulated in cancer cell lines and tumors. miPEP133 overexpression induced apoptosis in cancer cells and inhibited their migration and invasion. miPEP133 inhibited tumor growth in vivo. Low miPEP133 expression was an unfavorable prognostic marker associated with advanced metastatic NPC. Wild-type p53 but not mutant p53 induced miPEP133 expression. miPEP133 enhanced p53 transcriptional activation and miR-34a expression. miPEP133 localized in the mitochondria to interact with mitochondrial heat shock protein 70kD (HSPA9) and prevent HSPA9 from interacting with its binding partners, leading to the decrease of mitochondrial membrane potential and mitochondrial mass. CONCLUSION: miPEP133 is a tumor suppressor localized in the mitochondria. It is a potential prognostic marker and therapeutic target for multiple types of cancers.

Biological Activity of Artificial Plant Peptides Corresponding to the Translational Products of Small ORFs in Primary miRNAs and Other Long "Non-Coding" RNAs.

Generally, lncPEPs (peptides encoded by long non-coding RNAs) have been identified in many plant species of several families and in some animal species. Importantly, molecular mechanisms of the miPEPs (peptides encoded by primary microRNAs, pri-miRNAs) are often poorly understood in different flowering plants. Requirement for the additional studies in these directions is highlighted by alternative findings concerning positive regulation of pri-miRNA/miRNA expression by synthetic miPEPs in plants. Further extensive studies are also needed to understand the full set of their roles in eukaryotic organisms. This review mainly aims to consider the available data on the regulatory functions of the synthetic miPEPs. Studies of chemically synthesized miPEPs and analyzing the fine molecular mechanisms of their functional activities are reviewed. Brief description of the studies to identify lncORFs (open reading frames of long non-coding RNAs) and the encoded protein products is also provided.

The Essentials on microRNA-Encoded Peptides from Plants to Animals.

Primary transcripts of microRNAs (pri-miRNAs) were initially defined as long non-coding RNAs that host miRNAs further processed by the microRNA processor complex. A few years ago, however, it was discovered in plants that pri-miRNAs actually contain functional open reading frames (sORFs) that translate into small peptides called miPEPs, for microRNA-encoded peptides. Initially detected in Arabidopsis thaliana and Medicago truncatula, recent studies have revealed the presence of miPEPs in other pri-miRNAs as well as in other species ranging from various plant species to animals. This suggests that miPEP numbers remain largely underestimated and that they could be a common signature of pri-miRNAs. Here we present the most recent advances in miPEPs research and discuss how their discovery has broadened our vision of the regulation of gene expression by miRNAs, and how miPEPs could be interesting tools in sustainable agriculture or the treatment of certain human diseases.

Plant microProteins and miPEPs: Small molecules with much bigger roles.

The plant science community has identified various regulatory components involved in gene expression. With the advancement of approaches and technologies, new layers of gene regulation have been identified, which play essential roles in fine-tuning biological processes. In this area, recently, small peptides emerged as key regulators in gene regulation to control developmental and physiological processes in plants. Various small peptides have also been identified and characterized to elucidate their roles. A class of small peptides, microProteins (miPs), have been shown to contain at least a protein-protein interaction domain with the potential to regulate multi-domain proteins by becoming a part of protein complexes. Recent studies suggest that some pri-miRNAs encode peptides (miPEPs), which are essential components in plant growth and development. This review provides updates about these small peptides, in general, summarizing their potential role in gene regulation and possible mechanism(s) in plants. We also propose that in-depth research on newly identified plant peptides in crops help to provide solutions enabling sustainable agriculture and food production.

Case report: Rare novel MIPEP compound heterozygous variants presenting with hypertrophic cardiomyopathy, severe lactic acidosis and hypotonia in a Chinese infant.

Background: Mitochondrial intermediate peptidase, encoded by the MIPEP gene, is involved in the processing of precursor mitochondrial proteins related to oxidative phosphorylation. Only a few studies have shown that mutations in MIPEP can cause combined oxidative phosphorylation deficiency-31 (COXPD31), an autosomal recessive multisystem disorder associated with mitochondrial dysfunction. We report herein a rare case of an 8-month-old boy in China with hypertrophic cardiomyopathy (HCM), severe lactic acidosis, and hypotonia caused by novel MIPEP compound heterozygous variants. Methods: Trio-whole-exome sequencing and copy number variation sequencing were performed to identify mutated genetic loci. Sanger sequencing and quantitative real-time PCR were used to validate the candidate single nucleotide variants and copy number variants, respectively. Results: The proband was an 8-month-old boy with HCM, severe lactic acidosis, and hypotonia who died 2 months after his first admission. Two novel compound heterozygous variants, c.1081T > A (p. Tyr361Asn) and a whole deletion (Ex1-19 del), were found in the MIPEP gene, which were inherited from his healthy parents respectively. Additionally, his mitochondria DNA copy number was significantly reduced. Conclusion: We are the first to report a patient with rare MIPEP variants in China. Our findings expand the mutation spectrum of MIPEP, and provide insights into the genotype-phenotype relationship in COXPD31.

Drosophila primary microRNA-8 encodes a microRNA-encoded peptide acting in parallel of miR-8.

BACKGROUND: Recent genome-wide studies of many species reveal the existence of a myriad of RNAs differing in size, coding potential and function. Among these are the long non-coding RNAs, some of them producing functional small peptides via the translation of short ORFs. It now appears that any kind of RNA presumably has a potential to encode small peptides. Accordingly, our team recently discovered that plant primary transcripts of microRNAs (pri-miRs) produce small regulatory peptides (miPEPs) involved in auto-regulatory feedback loops enhancing their cognate microRNA expression which in turn controls plant development. Here we investigate whether this regulatory feedback loop is present in Drosophila melanogaster. RESULTS: We perform a survey of ribosome profiling data and reveal that many pri-miRNAs exhibit ribosome translation marks. Focusing on miR-8, we show that pri-miR-8 can produce a miPEP-8. Functional assays performed in Drosophila reveal that miPEP-8 affects development when overexpressed or knocked down. Combining genetic and molecular approaches as well as genome-wide transcriptomic analyses, we show that miR-8 expression is independent of miPEP-8 activity and that miPEP-8 acts in parallel to miR-8 to regulate the expression of hundreds of genes. CONCLUSION: Taken together, these results reveal that several Drosophila pri-miRs exhibit translation potential. Contrasting with the mechanism described in plants, these data shed light on the function of yet undescribed primary-microRNA-encoded peptides in Drosophila and their regulatory potential on genome expression.