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Cluster of differentiation 24 (CD24) is a cell surface glycoprotein, which is largely present on hematopoietic cells and many types of solid tumor cells. CD24 is known to be involved in a wide range of downstream signaling pathways and neural development, yet the underlying mechanisms are poorly understood. Moreover, its production correlates with poor cancer prognosis, and targeting of CD24 with different antibodies has been shown to inhibit disease progression. Nucleic acid aptamers are oligonucleotides that are selected from random DNA or RNA libraries for high affinity and specific binding to a certain target. Thus, they can be used as an alternative to antibodies. To gain an insight on CD24 role and its interaction partners, we performed several SELEX (systematic evolution of ligands by exponential enrichment) experiments to select CD24-specfiic DNA aptamers. We found that the cell-SELEX approach was the most useful and that using HT-29 cell line presenting CD24 along with CD24 knockdown HT-29 cells has selected six aptamers. For the selected aptamers, we determined dissociation constants in the nanomolar range (18-709 nM) using flow cytometry. These aptamers can be applied as diagnostic tools to track cancer progression and bear a potential for therapeutic use for inhibiting signaling pathways that promote the metastatic process.

To survive under conditions of stress, such as nutrient deprivation, bacterial 70S ribosomes dimerize to form hibernating 100S particles1. In γ-proteobacteria, such as Escherichia coli, 100S formation requires the ribosome modulation factor (RMF) and the hibernation promoting factor (HPF)2-4. Although structures of E. coli 100S particles have been reported5,6, the low resolution (18-38 Å) prevented the mechanism of ribosome inactivation and dimerization to be fully elucidated. Here we present single particle cryo-electron microscopy structures of hibernating 70S and 100S particles isolated from stationary phase E. coli cells at 3.0-7.9 Å resolution, respectively. Preferred orientation bias for the complete 100S particle was overcome using tilting during data collection. The structures reveal the binding sites for HPF and RMF as well as the unexpected presence of deacylated E-site tRNA and ribosomal protein S1 in the 100S particle. HPF interacts with the anticodon-stem-loop of the E-tRNA and occludes the binding site for the mRNA as well as A- and P-site tRNAs. RMF stabilizes a compact conformation of S1, which together sequester the anti-Shine-Dalgarno (SD) sequence of the 16S ribosomal RNA (rRNA), thereby inhibiting translation initiation. At the dimerization interface, S1 and S2 form intersubunit bridges with S3 and S4 and the C-terminus of S2 probes the mRNA entrance channel of the symmetry related particle, thus suggesting that only translationally inactive ribosomes are prone to dimerization. The back-to-back 100S dimerization mediated by HPF and RMF is distinct from that observed previously in Gram-positive bacteria7-10 and reveals a unique function for S1 in ribosome dimerization and inactivation, rather than its canonical role in facilitating translation initiation.

The 3′-termini of tRNA are the point of amino acid linkage and thus crucial for their function in delivering amino acids to the ribosome and other enzymes. Therefore, to provide tRNA functionality, cells have to ensure the integrity of the 3′-terminal CCA-tail, which is generated during maturation by the 3′-trailer processing machinery and maintained by the CCA-adding enzyme. We developed a new tRNA sequencing method that is specifically tailored to assess the 3′-termini of E. coli tRNA. Intriguingly, we found a significant fraction of tRNAs with damaged CCA-tails under exponential growth conditions and, surprisingly, this fraction decreased upon transition into stationary phase. Interestingly, tRNAs bearing guanine as a discriminator base are generally unaffected by CCA-tail damage. In addition, we showed tRNA species-specific 3′-trailer processing patterns and reproduced in vitro findings on preferences of the maturation enzyme RNase T in vivo.

Scientific reports 9(1), 2465 (2019). doi:10.1038/s41598-019-39213-2, Misfolding and aggregation of prion protein (PrP) causes neurodegenerative diseases like Creutzfeldt-Jakob disease (CJD) and scrapie. Besides the consensus that spontaneous conversion of normal cellular PrPC into misfolded and aggregating PrPSc is the central event in prion disease, an alternative hypothesis suggests the generation of pathological PrPSc by rare translational frameshifting events in the octa-repeat domain of the PrP mRNA. Ribosomal frameshifting most commonly relies on a slippery site and an adjacent stable RNA structure to stall translating ribosome. Hence, it is crucial to unravel the secondary structure of the octa-repeat domain of PrP mRNA. Each of the five octa-repeats contains a motif (GGCGGUGGUGGCUGGG) which alone in vitro forms a G-quadruplex. Since the propensity of mRNA to form secondary structure depends on the sequence context, we set to determine the structure of the complete octa-repeat region. We assessed the structure of full-length octa-repeat domain of PrP mRNA using dynamic light scattering (DLS), small angle X-ray scattering (SAXS), circular dichroism (CD) spectroscopy and selective 2′-hydroxyl acylation analysis by primer extension (SHAPE). Our data show that the PrP octa-repeat mRNA forms stable A-helical hairpins with no evidence of G-quadruplex structure even in the presence of G-quadruplex stabilizing agents., Published by Macmillan Publishers Limited, part of Springer Nature, [London]

Misfolding and aggregation of prion protein (PrP) causes neurodegenerative diseases like Creutzfeldt-Jakob disease (CJD) and scrapie. Besides the consensus that spontaneous conversion of normal cellular PrP

tRNA maturation and quality control are crucial for proper functioning of these transcripts in translation. In several organisms, defective tRNAs were shown to be tagged by poly(A) or CCACCA tails and subsequently degraded by 3′-exonucleases. In a deep-sequencing analysis of tRNA 3′-ends, we detected the CCACCA tag also in Escherichia coli. However, this tag closely resembles several 3′-trailers of tRNA precursors targeted for maturation and not for degradation. Here, we investigate the ability of two important exonucleases, RNase R and RNase T, to distinguish tRNA precursors with a native 3′-trailer from tRNAs with a CCACCA tag. Our results show that the degrading enzyme RNase R breaks down both tRNAs primed for degradation as well as precursor transcripts, indicating that it is a rather nonspecific RNase. RNase T, a main processing exonuclease involved in trimming of 3′-trailers, is very inefficient in converting the CCACCA-tagged tRNA into a mature transcript. Hence, while both RNases compete for trailer-containing tRNA precursors, the inability of RNase T to process CCACCA tails ensures that defective tRNAs cannot reenter the functional tRNA pool, representing a safeguard to avoid detrimental effects of tRNAs with erroneous integrity on protein synthesis. Furthermore, these data indicate that the RNase T-mediated end turnover of the CCA sequence represents a means to deliver a tRNA to a repeated quality control performed by the CCA-adding enzyme. Hence, originally described as a futile side reaction, the tRNA end turnover seems to fulfill an important function in the maintenance of the tRNA pool in the cell.

Translation of mRNA into an amino acid sequence is mediated by ribosomes. tRNAs deliver amino acids to the growing polypeptide chain by pairing to the cognate codons of the mRNA. The concentration of cellular tRNAs is not uniform and varies upon development, disease and environmental stress. The different availability of tRNAs affects the ribosomal speed and hence protein production.

This protocol describes the knock-down of specific tRNA(s) in mammalian cells using short hairpin RNA (shRNA)-based RNA interference (RNAi). We have used this method successfully to silence specific tRNAs in in HeLa1 and N2a cells2. References 1. Kirchner et al. Alteration of Protein Function by a Silent Polymorphism Linked to tRNA Abundance. PLoS Biology. 2017, in press. 2. Girstmair et al. Depletion of Cognate Charged Transfer RNA Causes Translational Frameshifting within the Expanded CAG Stretch in Huntingtin. Cell Reports. 2013, 3(1):148-59. DOI: http://dx.doi.org/10.1016/j.celrep.2012.12.019

This protocol describes the microarray-based quantification of human tRNAs. The tRNA microarray consists of 40 full-length tDNA probes recognising all 54 nuclearly encoded human isoacceptor tRNAs1. The array has been shown to reliably distinguish tRNA isoacceptors with a sequence difference of more than 8 nucleotides. Deacetylated tRNAs are selectively labeled with a fluorescent oligonucleotide hairpin which is covalently attached to the single-stranded 3' NCCA overhang, a commen feature of all tRNAs2. For comparison, differently labeled tRNAs from two different conditions or cell/tissue types are labeled with the oligonucleotide hairpins with two different colors, mixed and hybridized onto the tRNA microarray. Ratios between the two different fluorescent signals are then used for relative comparison of tRNAs between the two samples. The assay has also be applied to other eukaryotic or prokaryotic organisms. For more information please contact Prof. Zoya Ignatova (University of Hamburg, Hamburg, Germany). However, this protocol only describes the quantification of human tRNAs. Example of the successful usage of the tRNA array can be found in Ref. 3 and 4. References 1. Dittmar KA,Goodenbour JM, Pan T. Tissue-specific differences in human transfer RNA expression. PLoS Genetics. 2006,2(12):e221. DOI: 10.1371/journal.pgen.0020221 2. Dittmar KA, Mobley EM, Radek AJ, Pan T. Exploring the Regulation of tRNA Distribution on the Genomic Scale. Journal of Molecular Biology. 2004, 337:31–47. DOI: 10.1016/j.jmb.2004.01.024 3. Kirchner et al. Alteration of Protein Function by a Silent Polymorphism Linked to tRNA Abundance. PLoS Biology. 2017, in press. 4.Czech A, Wende S, Mörl M, Pan T, Ignatova Z. Reversible and Rapid Transfer-RNA Deactivation as a Mechanism of Translational Repression in Stress. PLoS Genetics. 2013, 9(8):e1003767. DOI: http://dx.doi.org/10.1371/journal.pgen.1003767

SummaryHuntington disease (HD), a dominantly inherited neurodegenerative disorder caused by the expansion of a CAG-encoded polyglutamine (polyQ) repeat in huntingtin (Htt), displays a highly heterogeneous etiopathology and disease onset. Here, we show that the translation of expanded CAG repeats in mutant Htt exon 1 leads to a depletion of charged glutaminyl-transfer RNA (tRNA)Gln-CUG that pairs exclusively to the CAG codon. This results in translational frameshifting and the generation of various transframe-encoded species that differently modulate the conformational switch to nucleate fibrillization of the parental polyQ protein. Intriguingly, the frameshifting frequency varies strongly among different cell lines and is higher in cells with intrinsically lower concentrations of tRNAGln-CUG. The concentration of tRNAGln-CUG also differs among different brain areas in the mouse. We propose that translational frameshifting may act as a significant disease modifier that contributes to the cell-selective neurotoxicity and disease course heterogeneity of HD on both cellular and individual levels.

Mutations that alter the amino acid sequence are known to potentially exert deleterious effects on protein function, whereas substitutions of nucleotides without amino acid change are assumed to be neutral for the protein's functionality. However, cumulative evidence suggests that synonymous substitutions might also induce phenotypic variability by affecting splicing accuracy, translation fidelity, and conformation and function of proteins. tRNA isoacceptors mediate the translation of codons to amino acids, and asymmetric tRNA abundance causes variations in the rate of translation of each single triplet. Consequently, the effect of a silent point mutation in the coding region could be significant due to differential abundances of the cognate tRNA(s), emphasizing the importance of precise assessment of tRNA composition. Here, we provide an overview of the methods used to quantitatively determine the concentrations of tRNA species and discuss synonymous mutations in the context of tRNA composition of the cell, thus providing a new twist on the detrimental impact of the silent mutations.