Deletion of the gene for protein L27 from the E. coli chromosome results in severe defects in cell growth. This deficiency is corrected by the expression of wild-type (wt) protein L27 from a plasmid. Examination of strains expressing L27 variants truncated at the N terminus reveals that the absence of as few as three amino acids leads to a decrease in growth rate, an impairment in peptidyl transferase activity, and a sharp decline in the labeling of L27 from the 3' end of a photoreactive tRNA at the ribosomal P site. These findings suggest that the flexible N-terminal sequence of L27, which protrudes onto the interface of the bacterial 50S subunit, can reach the peptidyl transferase active site and contribute to its function, possibly by helping to correctly position tRNA substrates at the catalytic site.

Quantitative PCR (qPCR) remains the most widely used technique for gene expression evaluation. Obtaining reliable data using this method requires reference genes (RGs) with stable mRNA level under experimental conditions. This issue is especially crucial in cancer studies because each tumor has a unique molecular portrait. The Cancer Genome Atlas (TCGA) project provides RNA-Seq data for thousands of samples corresponding to dozens of cancers and presents the basis for assessment of the suitability of genes as reference ones for qPCR data normalization. Using TCGA RNA-Seq data and previously developed CrossHub tool, we evaluated mRNA level of 32 traditionally used RGs in 12 cancer types, including those of lung, breast, prostate, kidney, and colon. We developed an 11-component scoring system for the assessment of gene expression stability. Among the 32 genes, PUM1 was one of the most stably expressed in the majority of examined cancers, whereas GAPDH , which is widely used as a RG, showed significant mRNA level alterations in more than a half of cases. For each of 12 cancer types, we suggested a pair of genes that are the most suitable for use as reference ones. These genes are characterized by high expression stability and absence of correlation between their mRNA levels. Next, the scoring system was expanded with several features of a gene: mutation rate, number of transcript isoforms and pseudogenes, participation in cancer-related processes on the basis of Gene Ontology, and mentions in PubMed-indexed articles. All the genes covered by RNA-Seq data in TCGA were analyzed using the expanded scoring system that allowed us to reveal novel promising RGs for each examined cancer type and identify several “universal” pan-cancer RG candidates, including SF3A1, CIAO1 , and SFRS4 . The choice of RGs is the basis for precise gene expression evaluation by qPCR. Here, we suggested optimal pairs of traditionally used RGs for 12 cancer types and identified novel promising RGs that demonstrate high expression stability and other features of reliable and convenient RGs (high expression level, low mutation rate, non-involvement in cancer-related processes, single transcript isoform, and absence of pseudogenes).

Human accelerated region 1 (HAR1) is a short DNA region identified recently to have evolved the most rapidly among highly constrained regions since the divergence from our common ancestor with chimpanzee. It is transcribed as part of a noncoding RNA specifically expressed in the developing human neocortex. Employing a panoply of enzymatic and chemical probes, our analysis of HAR1 RNA proposed a secondary structure model differing from that published. Most surprisingly, we discovered that the substitutions between the chimpanzee and human sequences led the human HAR1 RNA to adopt a cloverleaf-like structure instead of an extended and unstable hairpin in the chimpanzee sequence. Thus, the rapid evolutionary changes resulted in a profound rearrangement of HAR1 RNA structure. Altogether, our results provide a structural context for elucidating HAR1 RNA function.

A significant need for reliable and accurate cancer diagnostics and prognosis compels the search for novel biomarkers that would be able to discriminate between indolent and aggressive tumors at the early stages of disease. The aim of this work was identification of potential diagnostic biomarkers for characterization of different types of prostate tumors. NotI-microarrays with 180 clones associated with chromosome 3 genes/loci were applied to determine genetic and epigenetic alterations in 33 prostate tumors. For 88 clones, aberrations were detected in more than 10% of tumors. The major types of alterations were DNA methylation and/or deletions. Frequent methylation of the discovered loci was confirmed by bisulfite sequencing on selective sampling of genes: FGF12, GATA2, and LMCD1. Three genes (BHLHE40, BCL6, and ITGA9) were tested for expression level alterations using qPCR, and downregulation associated with hypermethylation was shown in the majority of tumors. Based on these data, we proposed the set of potential biomarkers for detection of prostate cancer and discrimination between prostate tumors with different malignancy and aggressiveness: BHLHE40, FOXP1, LOC285205, ITGA9, CTDSPL, FGF12, LOC440944/SETD5, VHL, CLCN2, OSBPL10/ZNF860, LMCD1, FAM19A4, CAND2, MAP4, KY, and LRRC58. Moreover, we probabilistically estimated putative functional relations between the genes within each set using the network enrichment analysis.

Structure-specific ligands are convenient tools for the recognition, targeting or probing of non-canonical DNA structures. Porphyrin derivatives exhibit a preference for interaction with G-quadruplex (G4) structures over canonical duplex DNA and are able to cause photoinducible damage to nucleic acids. Here, we show that Zn(II) 5,10,15,20-tetrakis(N-carboxymethyl-4-pyridinium)porphyrin (ZnP1) interacts with different conformations of the telomeric sequence d(TAGGG(TTAGGG)3) at submicromolar concentrations without any detectible disturbance of the particular fold. Among different folds, potassium (3+1) hybrid G4-structure. reveal the highest affinity to ZnP1. The pattern of guanine oxidation is specific for each telomeric DNA conformation and may serve as an additional tool for probing the G4 topology. The potassium (3+1) and parallel G4 conformations are more susceptible to light-induced oxidation than the sodium G4 conformation or double helix of the telomeric DNA. The major products of the guanine modifications are spiroiminodihydantoin (Sp) and 8-oxoguanine (8-oxoG). ZnP1-induced oxidation of guanines results in the structural rearrangement of parallel and (3+1) G4 conformations yielding an antiparallel-like G4 conformation. The mechanism of the observed light-induced conformational changes is discussed.

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Non-Small Cell Lung Cancer (NSCLC) is responsible for the majority of deaths caused by cancer. Small C-terminal domain (CTD) phosphatases (SCP), CTDSP1, CTDSP2 and CTDSPL (CTDSPs) belong to SCP/CTDSP subfamily and are involved in many vital cellular processes and tumorigenesis. High similarity of their structures suggests similar functions. However their role in NSCLC remains insufficiently understood. For the first time we revealed the suppressor function of CTDSPs leading to a significant growth slowdown and senescence of A549 lung adenocarcinoma (ADC) cells in vitro. Their tumor-suppressive activity can be realized through increasing the proportion of the active form of Rb protein dephosphorylated at Ser807/811, Ser780, and Ser795 (P<0.05) thereby negatively regulating cancer cell proliferation. Moreover, we observed that a frequent (84%, 39/46) and highly concordant (Spearman’s rank correlation coefficient (rs) = 0.53–0.62, P≤0.01) down-regulation of CTDSPs and RB1 is characteristic of primary NSCLC samples (n=46). A clear difference in their mRNA levels was found between lung ADCs with and without lymph node metastases, but not in squamous cell carcinomas (SCCs) (P≤0.05). Based on The Cancer Genome Atlas (TCGA) data and the results obtained using the CrossHub tool, we suggest that the well-known oncogenic cluster miR-96/182/183 could be a common expression regulator of CTDSPs. Indeed, according to our qPCR, the expression of CTDSPs negatively correlates with these miRs, but positively correlates with their intronic miR-26a/b. Our results reflect functional association of CTDSP1, CTDSP2, and CTDSPL, expand knowledge about their suppressor properties through Rb dephosphorylation and provide new insights into the regulation of NSCLC growth.