MicroRNAs (miRNAs) are small non-coding RNAs which contribute to the regulation of many physiological and pathological processes. Conventionally, miRNAs perform their activity in the cytoplasm where they regulate gene expression by interacting in a sequence-specific manner with mature messenger RNAs. Recent studies point to the presence of mature miRNAs in the nucleus. This review summarizes current findings regarding the molecular activities of nuclear miRNAs. These molecules can regulate gene expression at the transcriptional level by directly binding DNA on the promoter or the enhancer of regulated genes. miRNAs recruit different protein complexes to these regions, resulting in activation or repression of transcription, through a number of molecular mechanisms. Hematopoiesis is presented as a paradigmatic biological process whereby nuclear miRNAs possess a relevant regulatory role. Nuclear miRNAs can influence gene expression by affecting nuclear mRNA processing and by regulating pri-miRNA maturation, thus impacting the biogenesis of miRNAs themselves. Overall, nuclear miRNAs are biologically active molecules that can be critical for the fine tuning of gene expression and deserve further studies in a number of physiological and pathological conditions.

Bazooka/Par-3 (Baz) is an evolutionarily conserved scaffold protein that functions as a master regulator for the establishment and maintenance of cell polarity in many different cell types. In the vast majority of published research papers Baz has been reported to localize at the cell cortex and at intercellular junctions. However, there have also been several reports showing localization and function of Baz at additional subcellular sites, in particular the nuclear envelope and the neuromuscular junction. In this study we have re-assessed the localization of Baz to these subcellular sites in a systematic manner. We used antibodies raised in different host animals against different epitopes of Baz for confocal imaging of Drosophila tissues. We tested the specificity of these antisera by mosaic analysis with null mutant baz alleles and tissue-specific RNAi against baz. In addition, we used a GFP-tagged gene trap line for Baz and a bacterial artificial chromosome (BAC) expressing GFP-tagged Baz under control of its endogenous promoter in a baz mutant background to compare the subcellular localization of the GFP-Baz fusion proteins to the staining with anti-Baz antisera. Together, these experiments did not provide evidence for specific localization of Baz to the nucleus or the neuromuscular junction.

BACKGROUND: Merkel Cell Carcinoma (MCC) is an aggressive skin cancer that is three times deadlier than melanoma. In 2008, it was found that 80% of MCC cases are caused by the genomic integration of a novel polyomavirus, Merkel Cell Polyomavirus (MCPyV), and the expression of its small and truncated large tumor antigens (ST and LT-t, respectively). MCPyV belongs to a family of human polyomaviruses; however, it is the only one with a clear association to cancer.
METHODS: To investigate the role and mechanisms of various polyomavirus tumor antigens in cellular transformation, Rat-2 and 293A cells were transduced with pLENTI MCPyV LT-t, MCPyV ST, TSPyV ST, HPyV7 ST, or empty pLENTI and assessed through multiple transformation assays, and subcellular fractionations. One-way ANOVA tests were used to assess statistical significance.
RESULTS: Soft agar, proliferation, doubling time, glucose uptake, and serum dependence assays confirmed ST to be the dominant transforming protein of MCPyV. Furthermore, it was found that MCPyV ST is uniquely transforming, as the ST antigens of other non-oncogenic human polyomaviruses such as Trichodysplasia Spinulosa-Associated Polyomavirus (TSPyV) and Human Polyomavirus 7 (HPyV7) were not transforming when similarly assessed. Identification of structural dissimilarities between transforming and non-transforming tumor antigens revealed that the uniquely transforming domain(s) of MCPyV ST are likely located within the structurally dissimilar loops of the MCPyV ST unique region. Of all known MCPyV ST cellular interactors, 62% are exclusively or transiently nuclear, suggesting that MCPyV ST localizes to the nucleus despite the absence of a canonical nuclear localization signal. Indeed, subcellular fractionations confirmed that MCPyV ST could achieve nuclear localization through a currently unknown, regulated mechanism independent of its small size, as HPyV7 and TSPyV ST proteins were incapable of nuclear translocation. Although nuclear localization was found to be important for several transforming properties of MCPyV ST, some properties were also performed by a cytoplasmic sequestered MCPyV ST, suggesting that MCPyV ST may perform different transforming functions in individual subcellular compartments.
CONCLUSIONS: Together, these data further elucidate the unique differences between MCPyV ST and other polyomavirus ST proteins necessary to understand MCPyV as the only known human oncogenic polyomavirus.

RAMOSA1 (RA1) is a Cys2-His2-type (C2H2) zinc finger transcription factor that controls plant meristem fate and identity and has played an important role in maize domestication. Despite its importance, the origin of RA1 is unknown, and the evolution in plants is only partially understood. In this paper, we present a well-resolved phylogeny based on 73 amino acid sequences from 48 embryophyte species. The recovered tree topology indicates that, during grass evolution, RA1 arose from two consecutive SUPERMAN duplications, resulting in three distinct grass sequence lineages: RA1-like A, RA1-like B, and RA1; however, most of these copies have unknown functions. Our findings indicate that RA1 and RA1-like play roles in the nucleus despite lacking a traditional nuclear localization signal. Here, we report that copies diversified their coding region and, with it, their protein structure, suggesting different patterns of DNA binding and protein-protein interaction. In addition, each of the retained copies diversified regulatory elements along their promoter regions, indicating differences in their upstream regulation. Taken together, the evidence indicates that the RA1 and RA1-like gene families in grasses underwent subfunctionalization and neofunctionalization enabled by gene duplication.

Huntington\'s disease (HD) is an autosomal dominant neurodegenerative disease caused by CAG repeat expansion in the first exon of the huntingtin gene (HTT). Oligonucleotide therapeutics, such as short interfering RNA (siRNA), reduce levels of huntingtin mRNA and protein in vivo and are considered a viable therapeutic strategy. However, the extent to which they silence HTT mRNA in the nucleus is not established. We synthesized siRNA cross-reactive to mouse (wild-type) Htt and human (mutant) HTT in a di-valent scaffold and delivered to two mouse models of HD. In both models, di-valent siRNA sustained lowering of wild-type Htt, but not mutant HTT mRNA expression in striatum and cortex. Near-complete silencing of both mutant HTT protein and wild-type Htt protein was observed in both models. Subsequent fluorescent in situ hybridization (FISH) analysis shows that di-valent siRNA acts predominantly on cytoplasmic mutant HTT transcripts, leaving clustered mutant HTT transcripts in the nucleus largely intact in treated HD mouse brains. The observed differences between mRNA and protein levels, exaggerated in the case of extended repeats, might apply to other repeat-associated neurological disorders.

The AMPK/SNF1 pathway governs energy balance in eukaryotic cells, notably influencing glucose de-repression. In S. cerevisiae, Snf1 is phosphorylated and hence activated upon glucose depletion. This activation is required but is not sufficient for mediating glucose de-repression, indicating further glucose-dependent regulation mechanisms. Employing fluorescence recovery after photobleaching (FRAP) in conjunction with non-linear mixed effects modelling, we explore the spatial dynamics of Snf1 as well as the relationship between Snf1 phosphorylation and its target Mig1 controlled by hexose sugars. Our results suggest that inactivation of Snf1 modulates Mig1 localization and that the kinetic of Snf1 localization to the nucleus is modulated by the presence of non-fermentable carbon sources. Our data offer insight into the true complexity of regulation of this central signaling pathway in orchestrating cellular responses to fluctuating environmental cues. These insights not only expand our understanding of glucose homeostasis but also pave the way for further studies evaluating the importance of Snf1 localization in relation to its phosphorylation state and regulation of downstream targets.

Viruses infecting bacteria (bacteriophages) represent the most abundant viral particles in the human body. They participate in the control of the human-associated bacterial communities and play an important role in the dissemination of virulence genes. Here, we present the identification of a new filamentous single-stranded DNA phage of the family Inoviridae, named Ralstonia Inoviridae Phage 1 (RIP1), in the human blood. Metagenomics and PCR analyses detected the RIP1 genome in blood serum, in the absence of concomitant bacterial infection or contamination, suggesting inovirus persistence in the human blood. Finally, we have experimentally demonstrated that the RIP1-encoded rolling circle replication initiation protein and serine integrase have functional nuclear localization signals and upon expression in eukaryotic cells both proteins were translocated into the nucleus. This observation adds to the growing body of data suggesting that phages could have an overlooked impact on the evolution of eukaryotic cells.

The troponin complex-consisting of three subunits: troponin C (TnC), cardiac troponin I (cTnI) and cardiac troponin T (cTnT)-plays a key role in the regulation of myocardial contraction. Troponins are preferentially localized in the cytoplasm and bind to myofibrils. However, numerous, albeit scattered, studies have shown the presence of troponins in the nuclei of muscle cells. There is increasing evidence that the nuclear localization of troponins may be functionally important, making troponins an important nuclear player in the pathogenesis of various diseases including cancer and myopathies. Further studies in this area could potentially lead to the development of treatments for certain pathologies. In this review, we collected and discussed recent data on the properties of non-canonically localized cardiac troponins, the molecular mechanisms leading to this non-canonical localization, and the possible functions or pathological effects of these non-canonically localized troponins.

Hepatocyte nuclear factor-4 alpha (HNF-4A) regulates genes with roles in glucose metabolism and β-cell development. Although pathogenic HNF4A variants are commonly associated with maturity-onset diabetes of the young (MODY1; HNF4A-MODY), rare phenotypes also include hyperinsulinemic hypoglycemia, renal Fanconi syndrome and liver disease. While the association of rare functionally damaging HNF1A variants with HNF1A-MODY and type 2 diabetes is well established owing to robust functional assays, the impact of HNF4A variants on HNF-4A transactivation in tissues including the liver and kidney is less known, due to lack of similar assays. Our aim was to investigate the functional effects of seven HNF4A variants, located in the HNF-4A DNA binding domain and associated with different clinical phenotypes, by various functional assays and cell lines (transactivation, DNA binding, protein expression, nuclear localization) and in silico protein structure analyses. Variants R85W, S87N and R89W demonstrated reduced DNA binding to the consensus HNF-4A binding elements in the HNF1A promoter (35, 13 and 9%, respectively) and the G6PC promoter (R85W ~10%). While reduced transactivation on the G6PC promoter in HepG2 cells was shown for S87N (33%), R89W (65%) and R136W (35%), increased transactivation by R85W and R85Q was confirmed using several combinations of target promoters and cell lines. R89W showed reduced nuclear levels. In silico analyses supported variant induced structural impact. Our study indicates that cell line specific functional investigations are important to better understand HNF4A-MODY genotype-phenotype correlations, as our data supports ACMG/AMP interpretations of loss-of-function variants and propose assay-specific HNF4A control variants for future functional investigations.

Planar cell polarity (PCP) proteins coordinate tissue morphogenesis by governing cell patterning and polarity. Asymmetrically localized on the plasma membrane of cells, transmembrane PCP proteins are trafficked by endocytosis, suggesting they may have intracellular functions that are dependent or independent of their extracellular role, but whether these functions extend to transcriptional control remains unknown. Here, we show the nuclear localization of transmembrane, PCP protein, VANGL2, in the HCC1569 breast cancer cell line, and in undifferentiated, but not differentiated, HC11 cells that serve as a model for mammary lactogenic differentiation. The loss of Vangl2 function results in upregulation of pathways related to STAT5 signaling. We identify DNA binding sites and a nuclear localization signal in VANGL2, and use CUT&RUN to demonstrate recruitment of VANGL2 to specific DNA binding motifs, including one in the Stat5a promoter. Knockdown (KD) of Vangl2 in HC11 cells and primary mammary organoids results in upregulation of Stat5a, Ccnd1 and Csn2, larger acini and organoids, and precocious differentiation; phenotypes are rescued by overexpression of Vangl2, but not Vangl2ΔNLS. Together, these results advance a paradigm whereby PCP proteins coordinate tissue morphogenesis by keeping transcriptional programs governing differentiation in check.