Chemical probing for examining the structure of modified RNAs and ligand binding to RNA.

Among different RNA modifications, the helix 69 (H69) region of the bacterial ribosomal RNA (rRNA) contains three pseudouridines (Ψs). H69 is functionally important due to its location in the heart of the ribosome. Several structural and functional studies have shown the importance of Ψ modifications in influencing the H69 conformation as well as maintaining key interactions in the ribosome during protein synthesis. Therefore, a need exists to understand the influence of modified nucleosides on conformational dynamics of the ribosome under solution conditions that mimic the cellular environment. In this review on chemical probing, we provide detailed protocols for the use of dimethyl sulfate (DMS) to examine H69 conformational states and the influence of Ψ modifications under varying solution conditions in the context of both ribosomal subunits and full ribosomes. The use of DMS footprinting to study the binding of aminoglycosides to the H69 region of bacterial rRNA as a potential antibiotic target will also be discussed. As highlighted in this work, DMS probing and footprinting are versatile techniques that can be used to gain important insight into RNA local structure and RNA-ligand interactions, respectively.

Author Correction: Meikin is a conserved regulator of meiosis-I-specific kinetochore function.

An Amendment to this Article has been published and is linked from the HTML version of this paper.

Pseudouridine modifications influence binding of aminoglycosides to helix 69 of bacterial ribosomes.

Development of antibiotics that target new regions of functionality is a possible way to overcome antibiotic resistance. In this study, the interactions of aminoglycoside antibiotics with helix 69 of the E. coli 23S rRNA in the context of complete 70S ribosomes or the isolated 50S subunit were investigated by using chemical probing and footprinting analysis. Helix 69 is a dynamic RNA motif that plays major roles in bacterial ribosome activity. Neomycin, paromomycin, and gentamicin interact with the stem region of helix 69 in complete 70S ribosomes, but have diminished binding to the isolated 50S subunit. Pseudouridine modifications in helix 69 were shown to impact the aminoglycoside interactions. These results suggest a requirement for a specific conformational state of helix 69 for efficient aminoglycoside binding, and imply that this motif may be a suitable target for mechanism-based therapeutics.

Live Imaging of Intracellular Dynamics During Meiotic Maturation in Mouse Oocytes.

Fluorescence live imaging is a powerful approach to study intracellular dynamics during cellular events such as cell division. By applying automated confocal live imaging to mouse oocytes, in which meiotic maturation can be induced in vitro after the introduction of fluorescent proteins through microinjection, the meiotic dynamics of intracellular structures, such as chromosomes, can be monitored at high resolution. A combination of this method with approaches for the perturbation of specific proteins opens up opportunities for understanding the molecular and intracellular basis of mammalian meiosis.

DNA damage response during mouse oocyte maturation.

Because low levels of DNA double strand breaks (DSBs) appear not to activate the ATM-mediated prophase I checkpoint in full-grown oocytes, there may exist mechanisms to protect chromosome integrity during meiotic maturation. Using live imaging we demonstrate that low levels of DSBs induced by the radiomimetic drug Neocarzinostatin (NCS) increase the incidence of chromosome fragments and lagging chromosomes but do not lead to APC/C activation and anaphase onset delay. The number of DSBs, represented by γH2AX foci, significantly decreases between prophase I and metaphase II in both control and NCS-treated oocytes. Transient treatment with NCS increases >2-fold the number of DSBs in prophase I oocytes, but less than 30% of these oocytes enter anaphase with segregation errors. MRE11, but not ATM, is essential to detect DSBs in prophase I and is involved in H2AX phosphorylation during metaphase I. Inhibiting MRE11 by mirin during meiotic maturation results in anaphase bridges and also increases the number of γH2AX foci in metaphase II.  Compromised DNA integrity in mirin-treated oocytes indicates a role for MRE11 in chromosome integrity during meiotic maturation.

Bivalent separation into univalents precedes age-related meiosis I errors in oocytes.

The frequency of chromosome segregation errors during meiosis I (MI) in oocytes increases with age. The two-hit model suggests that errors are caused by the combination of a first hit that creates susceptible crossover configurations and a second hit comprising an age-related reduction in chromosome cohesion. This model predicts an age-related increase in univalents, but direct evidence of this phenomenon as a major cause of segregation errors has been lacking. Here, we provide the first live analysis of single chromosomes undergoing segregation errors during MI in the oocytes of naturally aged mice. Chromosome tracking reveals that 80% of the errors are preceded by bivalent separation into univalents. The set of the univalents is biased towards balanced and unbalanced predivision of sister chromatids during MI. Moreover, we find univalents predisposed to predivision in human oocytes. This study defines premature bivalent separation into univalents as the primary defect responsible for age-related aneuploidy.

Meikin is a conserved regulator of meiosis-I-specific kinetochore function.

The kinetochore is the crucial apparatus regulating chromosome segregation in mitosis and meiosis. Particularly in meiosis I, unlike in mitosis, sister kinetochores are captured by microtubules emanating from the same spindle pole (mono-orientation) and centromeric cohesion mediated by cohesin is protected in the following anaphase. Although meiotic kinetochore factors have been identified only in budding and fission yeasts, these molecules and their functions are thought to have diverged earlier. Therefore, a conserved mechanism for meiotic kinetochore regulation remains elusive. Here we have identified in mouse a meiosis-specific kinetochore factor that we termed MEIKIN, which functions in meiosis I but not in meiosis II or mitosis. MEIKIN plays a crucial role in both mono-orientation and centromeric cohesion protection, partly by stabilizing the localization of the cohesin protector shugoshin. These functions are mediated mainly by the activity of Polo-like kinase PLK1, which is enriched to kinetochores in a MEIKIN-dependent manner. Our integrative analysis indicates that the long-awaited key regulator of meiotic kinetochore function is Meikin, which is conserved from yeasts to humans.

Role of pseudouridine in structural rearrangements of helix 69 during bacterial ribosome assembly.

As part of the central core domain of the ribosome, helix 69 of 23S rRNA participates in an important intersubunit bridge and contacts several protein translation factors. Helix 69 is believed to play key roles in protein synthesis. Even though high-resolution crystal structures of the ribosome exist, the solution dynamics and roles of individual nucleotides in H69 are still not well-defined. To better understand the influence of modified nucleotides, specifically pseudouridine, on the multiple conformational states of helix 69 in the context of 50S subunits and 70S ribosomes, chemical probing analyses were performed on wild-type and pseudouridine-deficient bacterial ribosomes. Local structural rearrangements of helix 69 upon ribosomal subunit association and interactions with its partner, helix 44 of 16S rRNA, are observed. The helix 69 conformational states are also magnesium-dependent. The probing data presented in this study provide insight into the functional role of helix 69 dynamics and regulation of these conformational states by post-transcriptional pseudouridine modification.

Probing conformational states of modified helix 69 in 50S ribosomes.

The movement of the small ribosomal subunit (30S) relative to the large ribosomal subunit (50S) during translation is widely known, but many molecular details and roles of rRNA and proteins in this process are still undefined, especially in solution models. The functional relationship of modified nucleotides to ribosome activity is one such enigma. To better understand ribosome dynamics and the influence of modified nucleotides on such processes, the focus of this work was helix 69 of 23S rRNA, which contains three pseudouridine residues in its loop region. Ribosome probing experiments with dimethylsulfate revealed that specific base accessibilities and individual nucleotide conformations in helix 69 are influenced differently by pH, temperature, magnesium, and the presence of pseudouridine modifications.

Antitumor activity of sequence-specific alkylating agents: pyrolle-imidazole CBI conjugates with indole linker.

DNA-targeting agents, including cisplatin, bleomycin and mitomycin C, are used routinely in cancer treatments. However, these drugs are extremely toxic, attacking normal cells and causing severe side effects. One important question to consider in designing anticancer agents is whether the introduction of sequence selectivity to DNA-targeting agents can improve their efficacy as anticancer agents. In the present study, the growth inhibition activities of an indole-seco 1,2,9,9a-tetrahydrocyclopropa[1,2-c]benz[1,2-e]indol-4-one (CBI) (1) and five conjugates with hairpin pyrrole-imidazole polyamides (2-6), which have different sequence specificities for DNA alkylation, were compared using 10 different cell lines. The average values of -log GI50 (50% growth inhibition concentration) for compounds 1-6 against the 10 cell lines were 8.33, 8.56, 8.29, 8.04, 8.23 and 8.83, showing that all of these compounds strongly inhibit cell growth. Interestingly, each alkylating agent caused significantly different growth inhibition patterns with each cell line. In particular, the correlation coefficients between the -log GI50 of compound 1 and its conjugates 2-6 showed extremely low values (R<0). These results suggest that differences in the sequence specificity of DNA alkylation lead to marked differences in biological activity. Comparison of the correlation coefficients between compounds 6 and 7, with the same sequence specificity as 6, and MS-247, with sequence specificity different from 6, when used against a panel of 37 human cancer cell lines further confirmed the above hypothesis.