The balance of poly(U) polymerase activity ensures germline identity, survival and development in Caenorhabditis elegans.

Poly(U) polymerases (PUPs) catalyze 3' uridylation of mRNAs and small RNAs, a modification often correlating with decreased RNA stability. We have investigated the importance of three proteins with in vitro PUP activity, PUP-1/CDE-1, PUP-2 and PUP-3, in C. elegans germline development. Genetic analysis indicates that PUP-1/CDE-1 and PUP-2 are developmentally redundant under conditions of temperature stress during which they ensure germline viability and development. Multiple lines of evidence indicate that pup-1/-2 double mutant germ cells fail to maintain their identity as distinct from soma. Consistent with phenotypic data, PUP-1 and PUP-2 are expressed in embryonic germ cell precursors and throughout germline development. The developmental importance of PUP activity is presumably in regulating gene expression as both a direct and indirect consequence of modifying target RNAs. PUP-3 is significantly overexpressed in the pup-1/-2 germline, and loss of pup-3 function partially suppresses pup-1/-2 germline defects. We conclude that one major function of PUP-1/-2 is to limit PUP-3 expression. Overall, the balance of PUP-1, PUP-2 and PUP-3 activities appears to ensure proper germline development.

Progressive and Biased Divergent Evolution Underpins the Origin and Diversification of Peridinin Dinoflagellate Plastids.

Dinoflagellates are algae of tremendous importance to ecosystems and to public health. The cell biology and genome organization of dinoflagellate species is highly unusual. For example, the plastid genomes of peridinin-containing dinoflagellates encode only a minimal number of genes arranged on small elements termed "minicircles". Previous studies of peridinin plastid genes have found evidence for divergent sequence evolution, including extensive substitutions, novel insertions and deletions, and use of alternative translation initiation codons. Understanding the extent of this divergent evolution has been hampered by the lack of characterized peridinin plastid sequences. We have identified over 300 previously unannotated peridinin plastid mRNAs from published transcriptome projects, vastly increasing the number of sequences available. Using these data, we have produced a well-resolved phylogeny of peridinin plastid lineages, which uncovers several novel relationships within the dinoflagellates. This enables us to define changes to plastid sequences that occurred early in dinoflagellate evolution, and that have contributed to the subsequent diversification of individual dinoflagellate clades. We find that the origin of the peridinin dinoflagellates was specifically accompanied by elevations both in the overall number of substitutions that occurred on plastid sequences, and in the Ka/Ks ratio associated with plastid sequences, consistent with changes in selective pressure. These substitutions, alongside other changes, have accumulated progressively in individual peridinin plastid lineages. Throughout our entire dataset, we identify a persistent bias toward non-synonymous substitutions occurring on sequences encoding photosystem I subunits and stromal regions of peridinin plastid proteins, which may have underpinned the evolution of this unusual organelle.

Integration of plastids with their hosts: Lessons learned from dinoflagellates.

After their endosymbiotic acquisition, plastids become intimately connected with the biology of their host. For example, genes essential for plastid function may be relocated from the genomes of plastids to the host nucleus, and pathways may evolve within the host to support the plastid. In this review, we consider the different degrees of integration observed in dinoflagellates and their associated plastids, which have been acquired through multiple different endosymbiotic events. Most dinoflagellate species possess plastids that contain the pigment peridinin and show extreme reduction and integration with the host biology. In some species, these plastids have been replaced through serial endosymbiosis with plastids derived from a different phylogenetic derivation, of which some have become intimately connected with the biology of the host whereas others have not. We discuss in particular the evolution of the fucoxanthin-containing dinoflagellates, which have adapted pathways retained from the ancestral peridinin plastid symbiosis for transcript processing in their current, serially acquired plastids. Finally, we consider why such a diversity of different degrees of integration between host and plastid is observed in different dinoflagellates and how dinoflagellates may thus inform our broader understanding of plastid evolution and function.

Removal of protein S1 from Escherichia coli ribosomes without the use of affinity chromatography.

The paper reports an inexpensive and efficient procedure for the removal of protein S1 from E. coli ribosomes. It comprises incubation of ribosomes in a pyrimidine polyribonucleotide solution followed by centrifugation of the sample through a sucrose cushion. To avoid co-sedimentation of the S1-bound polypyrimidine with the ribosomes, its length should not exceed several hundred nucleotides. Unlike popular affinity chromatography through a poly(U) Sepharose or poly(U) cellulose column, the method tolerates limited polyribonucleotide degradation by eventual traces of ribonucleases, and can readily be incorporated into standard protocols for the isolation of ribosomes by centrifugation.

Broad and adaptable RNA structure recognition by the human interferon-induced tetratricopeptide repeat protein IFIT5.

Interferon (IFN) responses play key roles in cellular defense against pathogens. Highly expressed IFN-induced proteins with tetratricopeptide repeats (IFITs) are proposed to function as RNA binding proteins, but the RNA binding and discrimination specificities of IFIT proteins remain unclear. Here we show that human IFIT5 has comparable affinity for RNAs with diverse phosphate-containing 5'-ends, excluding the higher eukaryotic mRNA cap. Systematic mutagenesis revealed that sequence substitutions in IFIT5 can alternatively expand or introduce bias in protein binding to RNAs with 5' monophosphate, triphosphate, cap0 (triphosphate-bridged N7-methylguanosine), or cap1 (cap0 with RNA 2'-O-methylation). We defined the breadth of cellular ligands for IFIT5 by using a thermostable group II intron reverse transcriptase for RNA sequencing. We show that IFIT5 binds precursor and processed tRNAs, as well as other RNA polymerase III transcripts. Our findings establish the RNA recognition specificity of the human innate immune response protein IFIT5.

The first echinoderm poly-U-binding factor 60 kDa (PUF60) from sea cucumber (Stichopus monotuberculatus): Molecular characterization, inducible expression and involvement of apoptosis.

Poly-U-binding factor 60 kDa (PUF60), also known as Ro RNA binding protein (RoBPI) and FBP interacting repressor (FIR), is a multifunctional protein that is involved in a variety of nuclear processes including pre-mRNA splicing, apoptosis and transcription regulation. In this study, the first echinoderm PUF60 named StmPUF60 was identified from sea cucumber (Stichopus monotuberculatus). The StmPUF60 cDNA is 4503 bp in length, containing a 5'-untranslated region (UTR) of 34 bp, a 3'-UTR of 2963 bp and an open reading frame (ORF) of 1506 bp that encoding a protein of 501 amino acids with a deduced molecular weight of 54.15 kDa and a predicted isoelectric point of 5.15. The putative StmPUF60 protein possesses all the main characteristics of known PUF60 proteins, including two RNA recognition motifs (RRM1 and RRM2), a C-terminal PUMP domain and two conserved nucleic acid-binding ribonucleoprotein sequences (RNP1 and RNP2). For the gene structure, StmPUF60 contains nine exons separated by eight introns. In addition, the highest level of StmPUF60 mRNA expression was noticed in the gonad, followed by coelomocytes, intestine, respiratory tree and body wall. In in vivo experiments, the expression of StmPUF60 mRNA in coelomocytes and intestine was significantly up-regulated by lipopolysaccharides (LPS) challenge, suggesting that the sea cucumber PUF60 might play critical roles in the innate immune defense against bacterial infections. Moreover, we further confirmed that overexpressed StmPUF60 could induce apoptosis, and this function of StmPUF60 may be one of the innate immune defense mechanisms for sea cucumber against pathogen infections.

Poly(U) polymerase activity in Caenorhabditis elegans regulates abundance and tailing of sRNA and mRNA.

Terminal nucleotidyl transferases add nucleotides to the 3' end of RNA to modify their stability and function. In Caenorhabditis elegans, the terminal uridyltransferases/poly(U) polymerases PUP-1 (aka CID-1, CDE-1), PUP-2, and PUP-3 affect germline identity, survival, and development. Here, we identify small RNA (sRNA) and mRNA targets of these PUPs and of a fourth predicted poly(U) polymerase, F43E2.1/PUP-4. Using genetic and RNA sequencing approaches, we identify RNA targets of each PUP and the U-tail frequency and length of those targets. At the whole organism level, PUP-1 is responsible for most sRNA U-tailing, and other PUPs contribute to modifying discrete subsets of sRNAs. Moreover, expression of PUP-2, PUP-3, and especially PUP-4 limit uridylation on some sRNAs. The relationship between uridylation status and sRNA abundance suggests that U-tailing can have a negative or positive effect on abundance depending on context. sRNAs modified by PUP activity primarily target mRNAs that are ubiquitously expressed or most highly expressed in the germline. mRNA data obtained with a Nanopore-based method reveal that addition of U-tails to non-adenylated mRNA is substantially reduced in the absence of PUP-3. Overall, this work identifies PUP RNA targets, defines the effect of uridylation loss on RNA abundance, and reveals the complexity of PUP regulation in C. elegans development.

The Engineered Drug 3'UTRMYC1-18 Degrades the c-MYC-STAT5A/B-PD-L1 Complex In Vivo to Inhibit Metastatic Triple-Negative Breast Cancer.

c-MYC is overexpressed in 70% of human cancers, including triple-negative breast cancer (TNBC), yet there is no clinically approved drug that directly targets it. Here, we engineered the mRNA-stabilizing poly U sequences within the 3'UTR of c-MYC to specifically destabilize and promote the degradation of c-MYC transcripts. Interestingly, the engineered derivative outcompetes the endogenous overexpressed c-MYC mRNA, leading to reduced c-MYC mRNA and protein levels. The iron oxide nanocages (IO-nanocages) complexed with MYC-destabilizing constructs inhibited primary and metastatic tumors in mice bearing TNBC and significantly prolonged survival by degrading the c-MYC-STAT5A/B-PD-L1 complexes that drive c-MYC-positive TNBC. Taken together, we have described a novel therapy for c-MYC-driven TNBC and uncovered c-MYC-STAT5A/B-PD-L1 interaction as the target.

RNA-binding of Ru(II) complexes [Ru(phen)2(7-OCH3-dppz)]2+ and [Ru(phen)2(7-NO2-dppz)]2+: The former serves as a molecular "light switch" for poly(A)•poly(U).

To further determine the factors that affect the binding properties of ruthenium(II) polypyridine complexes with RNA duplex and to find excellent RNA-binding agents, the binding properties of ruthenium(II) complexes [Ru(phen)2(7-OCH3-dppz)]2+ (Ru1, phen = 1,10-phenan- throline, 7-OCH3-dppz = 7-methoxy-dipyrido-[3,2-a,2',3'-c]-phenazine) and [Ru(phen)2(7-NO2- dppz)]2+ (Ru2, 7-NO2-dppz = 7-nitro-dipyrido-[3,2-a,2',3'-c]-phenazine) with RNA poly(A)•poly(U) duplex have been investigated by spectroscopic methods and viscosity measurements in this work. The results show that complexes Ru1 and Ru2 bind to poly(A)•poly(U) through intercalation and the binding affinity between Ru2 and poly(A)•poly(U) is greater than that of Ru1. Thermal denaturation experiments suggest that both ruthenium(II) complexes exhibit a significant stabilizing effect on poly(A)•poly(U) duplex. Moreover, fluorescence emission spectra exhibit that, deviating from Ru2, Ru1 exhibits a "light switch" effect for poly(A)•poly(U). This effect can be observed by the naked eye under UV light and adjusted by pH, meaning that Ru1 may act as a reversible pH controlled molecular "light switch". The results obtained in this work will contribute to our understanding of the significant influence of the intercalative ligand substituent effect in the binding process of ruthenium(II) complexes with RNA duplex.

Integrative meta-analysis of gene expression profiles identifies FEN1 and ENDOU as potential diagnostic biomarkers for cervical squamous cell carcinoma.

Cervical carcinoma is a global public health burden. Given that it is usually asymptomatic at potentially curative stages, the development of clinically accurate tests is critical for early detection and individual risk stratification. The present study performed an integrative meta-analysis of the transcriptomes from 10 cervical carcinoma cohorts, with the aim of identifying biomarkers that are associated with malignant transformation of cervical epithelium, and establish their clinical applicability. From among the top ranked differentially expressed genes, flap structure-specific endonuclease 1 (FEN1) and poly (U)-specific endoribonuclease (ENDOU) were selected for further validation, and their clinical applicability was assessed using immunohistochemically stained microarrays comprising 110 tissue cores, using p16 and Ki67 staining as the comparator tests. The results demonstrated that FEN1 expression was significantly upregulated in 65% of tumor specimens (P=0.0001), with no detectable expression in the non-tumor tissues. Furthermore, its expression was significantly associated with Ki67 staining in tumor samples (P<0.0001), but no association was observed with p16 expression or the presence of human papilloma virus types 16/18, patient age, tumor grade or stage. FEN1 staining demonstrated lower sensitivity than p16 (69.3 vs. 96.8%) and Ki67 (69.3 vs. 76.3%); however, the specificity was identical to p16 and higher than that of Ki67 (100 vs. 71.4%).ENDOU staining was consistent with the microarray results, demonstrating 1% positivity in tumors and 40% positivity in non-tumor tissues. Gene set enrichment analysis of cervical tumors overexpressing FEN1 revealed its association with enhanced growth factor signaling, immune response inhibition and extracellular matrix remodeling, whereas tumors with low ENDOU expression exhibited inhibition of epithelial development and differentiation processes. Taken together, the results of the present study demonstrate the feasibility of the integrative meta-analysis approach to identify relevant biomarkers associated with cervical carcinogenesis. Thus, FEN1 and ENDOU may be useful diagnostic biomarkers for squamous cervical carcinoma. However, further studies are required to determine their diagnostic performance in larger patient cohorts and validate the results presented here.