Short ORF-encoded peptides (SEPs) are increasingly being identified as functional elements in various cellular processes. The current computational methods and experimental molecular biochemistry allow us to discover putative SEPs or micropeptides from proteogenomic datasets and experimentally validate them. Here, we identified a micropeptide produced from a putative long noncoding RNA (lncRNA) 1810058I24Rik which is downregulated in both human and murine myeloid cells exposed to lipopolysaccharide (LPS), as well as other TLR ligands and inflammatory cytokines. Analysis of lncRNA 1810058I24Rik subcellular localization revealed this transcript is localized in the cytosol, prompting us to evaluate its coding potential. In vitro translation with 35S-labeled methionine resulted in translation of a 47 amino acid micropeptide. Microscopy and subcellular fractionation studies in macrophages demonstrated endogenous expression of this peptide on the mitochondrion. We thus named this gene ‘Mitochondrial micropeptide-47 (Mm47)’. Functional studies using siRNA and Cripsr-cas9-mediated deletion in primary cells, showed that the transcriptional response downstream of TLR4 was not affected by Mm47 loss of function. In contrast, both the Crispr-cas9- and siRNA-targeted BMDM cells were compromised for Nlrp3 inflammasome responses. However, the primary macrophages derived from the Mm47 knockout mice do not require Mm47 for Nlrp3 activation, likely due to basal downregulation of a negative regulator microRNA of Nlrp3 called Mir-223. Notably, the Mm47-deficient mice are susceptible to influenza virus infection and succumb despite comparable antiviral and inflammatory response to wildtype mice. We hypothesize that the Mm47 deficiency may affect the antiviral resilience of mice due to secondary mitochondria dependent immunometabolic defect or failure of recovery from immune pathology, which warrants further investigation. This study therefore identifies a novel mitochondrial micropeptide Mm47 that is required for activation of the Nlrp3 inflammasome in cells and resistance to influenza virus infection. Broadly, this work highlights the presence of translatable ORFs is annotated noncoding RNA transcripts and underscores their importance in innate immunity and virus infection.

Pre-mRNA splicing is an important level in posttranscriptional gene regulation that is essential for accurate protein synthesis and generating protein diversity. The abundance of cryptic splice sites and long intronic DNA sequences makes their splicing a complex one. The identification of correct exons and introns needs additional information in the form of splicing regulatory elements (SREs) along with canonical splice signals. The interplay among these SREs and the trans factors (which bind to SREs) gives the identity to introns and exons which in turn leads to precise pre-mRNA splicing. Previous studies from our laboratory showed, that when expressed in mammalian cells from an expression vector, PDGF-B ORF was re-spliced at 4/5 exon junction with the downstream SV40 splice acceptor site in the vector. However, deletion of the 66-nt PDGF-B 3’ UTR region resulted in about 25% reduction in re-splicing. Sequence analysis of this region revealed presence of binding sites for splicing factors ASF/SF2 and SRp55, and an AU-rich element (ARE), mutation each of which affected re-splicing partially. In mammals, AREs are commonly found in the 3’UTR of mRNAs encoding proteins involved in diverse functions and are involved in selective mRNA degradation. Several ARE binding proteins are crucial for ARE’s function. Since mutation of the single ARE in the 3’UTR region altered the re-splicing efficiency, the role of AU-rich elements and ARE-binding proteins (AU-BPs) in modulation of splicing was investigated using siRNAs against AU-BPs, BRF1, hnRNPD, HuR, GAPDH and TTP. Down regulation of expression of these factors indeed affected the level of re-spliced product. We have studied the interactions between the full-length splicing factors (U1-70K and U2AF35) and the AU-BPs (BRF1, hnRNPD and HuR) as well as among the AU-BPs using three different assay methods: Yeast-two hybrid, co-immunoprecipitation and pull down assays. Our study has revealed that the BRF1 interacts with U1-70K and U2AF35 as well as the other AU-BPs hnRNPD and HuR but with different affinities. We have also analyzed the ability of AU-BPs to interact with SR proteins SRp20 and 9G8. We did find strong interaction of BRF1 with SRp20 and 9G8. Generation of a large number of nested deletion mutants of all the proteins allowed us to identify the interaction regions on the surface of BRF1, U1-70K, hnRNPD, U2AF35 and HuR. The results of Y2H analyses were further confirmed by pull down assay using purified interacting regions. It was found that a single region from aa 181-254 in BRF1 interacts with multiple partners i.e., splicing factors and the AU-BP hnRNPD. However, the RNA-binding zinc-finger domain from residue 120-181 independently interacts with HuR. Further, the multiple protein interacting region (MPIR) (aa 181-254) in BRF1 exhibits different affinities towards its interacting partners with that for U1-70K and hnRNPD being stronger than that for U2AF35 and HuR. This observation suggests that BRF1 activity can be modulated by interaction with different partners at different sites. U1-70K interacted only with BRF1 among the proteins tested in this study and this interaction appears to be RNA independent .This could have implications in splice site selection and RNA stability since BRF1 has been shown to promote RNA degradation. While the Arg/Glu-rich C-terminal region in U1-70K is sufficient for its interaction with BRF1, U2AF35 requires both the zinc-finger 2 and the arg/Gly/Ser-rich C-terminal regions for its association with BRF1. hnRNPD also interacts with multiple partners that include BRF1, HuR and U2AF35 using the N-terminal region that harbors a Ala-rich domain. The interaction of hnRNPD with HuR is RNA dependent while with BRF1 and U2AF35, it is RNA independentt. Further, its interaction with all the partners is equally strong. This suggests that hnRNPD could exert differential influence depending on the context of its interaction and abundance of the interacting partner. HuR, primarily known as an mRNA stabilizing factor, interacts with both BRF1 and hnRNPD with equal affinity involving the hinge region, the interaction with the former being RNA-independent and the later being RNA-dependent. This differential RNA-dependent and independent interactions with the two AU-BPs using a single interacting domain suggests a balancing act of HuR on the activities of BRF1 and hnRNPD. These interactions can further be differentially modulated by posttranslational modifications on one or all of the interacting partners depending on the physiological status of the cell. We have also analyzed the multiple protein complexes formed in absence of cellular RNA. Though we are unable to see direct protein-protein interaction between HuR and U1-70K in Yeast two hybrid analysis, we could detect the presence of U1-70K in HuR immunoprecipitate. It appears that U1-70K associates with HuR via BRF. We also detected the presence of HuR in U1-70K complexes which could be due to its association with BRF1. We are unable to find hnRNPD and U2AF35 in these complexes indicating that they may have been excluded. In anti-U2AF35 immunoprecipitates, we detected the presence of U1-70K as well as hnRNPD but no HuR. This may be due to RNase treatment as hnRNPD and HuR interactions are RNA dependent. Our findings that AU-rich elements in conjunction with AU-BPs function as intronic splicing modulators or enhancers, reveal hitherto unidentified new players in the poorly understood complex mechanisms that mediate alternative splicing. The possibility of dynamic nature of the interactions among splicing factors and AU-BPs mediated by post-translational modifications provide a basis for rapid cellular responses to changing environmental cues through generation of differentially spliced mRNAs and corresponding protein products that differ in their stability and hence their relative abundance. Our results also unfold enormous possibilities for future investigations on interactions among the many splicing factors and AU-BPs, and in understanding these complex interactions in modulation of pre-mRNA splicing, mRNA translation and degradation. The finding of coupling of AU-BPs to splicing machinery could further lead to better understanding of the mechanism of AU-BP-mediated targeting of mRNAs to processing bodies and ultimate degradation of the mRNAs.

Kürzlich wurde mithilfe von Ribo-seq Experimenten die Translation hunderter Mikroproteine in menschlichen Herzen entdeckt. Diese blieben zuvor aufgrund ihrer geringen Größe (< 100 Aminosäuren) unentdeckt, und ihre physiologische Rolle ist noch weitgehend unbekannt. Ziel dieser Promotionsarbeit ist es, potentielle Funktionen dieser neuartigen Mikroproteine zu entschlüsseln. Dabei sollen insbesondere die Aufklärung ihrer evolutionären Konservierungssignatur, subzellulären Lokalisierung und ihres Proteininteraktoms helfen. Die Konservierungsanalyse ergab, dass fast 90% der Mikroproteine nur in Primaten konserviert ist. Weiterhin konnte ich die Produktion von Mikroproteine in vitro und in vivo nachweisen, die subzelluläre Lokalisierung von 92 Mikroproteinen definieren, und Interaktionspartner für 60 Mikroproteine identifizieren. Dutzende dieser Mikroproteine lokalisieren in Mitochondrien. Dazu gehörte ein im Herzen angereichertes Mikroprotein, das aufgrund der Interaktions- und Lokalisationsdaten einen neuartigen Modulator der mitochondrialen Proteintranslation darstellen könnte. Der Interaktom-Screen zeigte außerdem, dass evolutionär junge Mikroproteine ähnliche Interaktionsfähigkeiten wie konservierte Kandidaten haben. Schließlich wurden kurze Sequenzmotive identifiziert, die Mikroprotein-Protein-Wechselwirkungen vermitteln, wodurch junge Mikroproteine mit zellulären Prozessen – wie z.B. Endozytose und Spleißen – in Verbindung gebracht werden konnten. Zusammenfassend wurde die Produktion vieler kleiner Proteine im menschlichen Herzen bestätigt, von denen die meisten lediglich in Primaten konserviert sind. Zusätzlich verknüpften umfangreiche Lokalisierungs- und Interaktionsdaten mehrere Mikroproteine mit Prozessen wie Spleißen, Endozytose und mitochondrialer Translation. Weitere Untersuchungen dieses zuvor verborgenen Teils des Herzproteoms werden zu einem besseren Verständnis von evolutionär jungen Proteinen und kardiologischen Prozessen beitragen. / Recently, the active translation of hundreds of previously unknown microproteins was detected using ribosome profiling on tissues of human hearts. They had remained undetected due to their small size (< 100 amino acids), and their physiological roles are still largely unknown. This dissertation aims to investigate these novel microproteins and validate their translation by independent methods. Particularly, elucidating their conservation signature, subcellular localization, and protein interactome shall aid in deciphering their potential biological role. Conservation analysis revealed that sequence conservation of almost 90% of microproteins was restricted to primates. I next confirmed microprotein production in vitro and in vivo by in vitro translation assays and mass spectrometry-based approaches, defined the subcellular localization of 92 microproteins, and identified significant interaction partners for 60 candidates. Dozens of these microproteins localized to the mitochondrion. These included a novel cardiac-enriched microprotein that may present a novel modulator of mitochondrial protein translation based on its interaction profile and subcellular localization. The interactome screen further revealed that evolutionarily young microproteins have similar interaction capacities to conserved candidates. Finally, it allowed identifying short linear motifs that may mediate microprotein-protein interactions and implicated several young microproteins in distinct cellular processes such as endocytosis and splicing. I conclude that many novel small proteins are produced in the human heart, most of which exhibit poor sequence conservation. I provide a substantial resource of microprotein localization and interaction data that links several to cellular processes such as splicing, endocytosis, and mitochondrial translation. Further investigation into this hidden part of the cardiac proteome will contribute to our understanding of recently evolved proteins and heart biology.