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Abstract Background Invasion is an important early step of cancer metastasis that is not well understood. Developing therapeutics to limit metastasis requires the identification and validation of candidate proteins necessary for invasion and migration. Methods We developed a functional proteomic screen to identify mediators of tumor cell invasion. This screen couples Fluorophore Assisted Light Inactivation (FALI) to a scFv antibody library to systematically inactivate surface proteins expressed by human fibrosarcoma cells followed by a high-throughput assessment of transwell invasion. Results Using this screen, we have identified CD155 (the poliovirus receptor) as a mediator of tumor cell invasion through its role in migration. Knockdown of CD155 by FALI or by RNAi resulted in a significant decrease in transwell migration of HT1080 fibrosarcoma cells towards a serum chemoattractant. CD155 was found to be highly expressed in multiple cancer cell lines and primary tumors including glioblastoma (GBM). Knockdown of CD155 also decreased migration of U87MG GBM cells. CD155 is recruited to the leading edge of migrating cells where it colocalizes with actin and αv-integrin, known mediators of motility and adhesion. Knockdown of CD155 also altered cellular morphology, resulting in cells that were larger and more elongated than controls when plated on a Matrigel substrate. Conclusion These results implicate a role for CD155 in mediating tumor cell invasion and migration and suggest that CD155 may contribute to tumorigenesis.

Background: Human papillomavirus (HPV) vaccine hesitancy is a growing concern in the United States, yet understudied among racial/ethnic minority parents. We conducted qualitative research to understand parental HPV vaccine hesitancy and inform community-specific, multilevel approaches to improve HPV vaccination among diverse populations in Los Angeles., Methods: We recruited American Indian/Alaska Native (AI/AN), Hispanic/Latino/a (HL) and Chinese parents of unvaccinated children (9-17 years) from low-HPV vaccine uptake regions in Los Angeles for virtual focus groups (FGs). FGs were conducted in English (2), Mandarin (1), and Spanish (1) between June-August 2021. One English FG was with AI/AN-identifying parents. FGs prompted discussions about vaccine knowledge, sources of information/hesitancy, logistical barriers and interpersonal, healthcare and community interactions regarding HPV vaccination. Guided by the social-ecological model, we identified multilevel emergent themes related to HPV vaccination., Results: Parents (n = 20) in all FGs reported exposure to HPV vaccine information from the internet and other sources, including in-language media (Mandarin) and health care providers (Spanish). All FGs expressed confusion around the vaccine and had encountered HPV vaccine misinformation. FGs experienced challenges navigating relationships with children, providers, and friends/family for HPV vaccine decision-making. At the community-level, historical events contributed to mistrust (e.g., forced community displacement [AI/AN]). At the societal-level, transportation, and work schedules (Spanish, AI/AN) were barriers to vaccination. Medical mistrust contributed to HPV vaccine hesitancy across the analysis levels., Conclusion: Our findings highlight the importance of multilevel influences on parental HPV vaccine hesitancy and decision-making and the need for community-specific messaging to combat medical mistrust and other barriers to HPV vaccination among racial/ethnic minority communities., (© 2023. The Author(s).)

Ribosome production is an essential cellular process involving a plethora of trans-acting factors, such as nucleases, methyltransferases, RNA helicases and kinases that catalyse key maturation steps. Precise temporal and spatial regulation of such enzymes is essential to ensure accurate and efficient subunit assembly. Here, we focus on the maturation of the 3' end of the 18S rRNA in human cells. We reveal that human RIO2 is an active kinase that phosphorylates both itself and the rRNA methyltransferase DIM1 in vitro. In contrast to yeast, our data confirm that human DIM1 predominantly acts in the nucleus and we further demonstrate that the 21S pre-rRNA is the main target for DIM1-catalysed methylation. We show that the PIN domain of the endonuclease NOB1 is required for site 3 cleavage, while the zinc ribbon domain is essential for pre-40S recruitment. Furthermore, we also demonstrate that NOB1, PNO1 and DIM1 bind to a region of the pre-rRNA encompassing the 3' end of 18S and the start of ITS1, in vitro. Interestingly, NOB1 is present in the cell at higher levels than other pre-40S factors. We provide evidence that NOB1 is multimeric within the cell and show that NOB1 multimerisation is lost when ribosome biogenesis is blocked. Taken together, our data indicate a dynamic interplay of key factors associated with the 3' end of the 18S rRNA during human pre-40S biogenesis and highlight potential mechanisms by which this process can be regulated.

Ribosome synthesis is an essential cellular process, and perturbation of human ribosome production is linked to cancer and genetic diseases termed ribosomopathies. During their assembly, pre-ribosomal particles undergo numerous structural rearrangements, which establish the architecture present in mature complexes and serve as key checkpoints, ensuring the fidelity of ribosome biogenesis. RNA helicases are essential mediators of such remodelling events and here, we demonstrate that the DEAH-box RNA helicase DHX37 is required for maturation of the small ribosomal subunit in human cells. Our data reveal that the presence of DHX37 in early pre-ribosomal particles is monitored by a quality control pathway and that failure to recruit DHX37 leads to pre-rRNA degradation. Using an in vivo crosslinking approach, we show that DHX37 binds directly to the U3 small nucleolar RNA (snoRNA) and demonstrate that the catalytic activity of the helicase is required for dissociation of the U3 snoRNA from pre-ribosomal complexes. This is an important event during ribosome assembly as it enables formation of the central pseudoknot structure of the small ribosomal subunit. We identify UTP14A as a direct interaction partner of DHX37 and our data suggest that UTP14A can act as a cofactor that stimulates the activity of the helicase in the context of U3 snoRNA release.

Production of eukaryotic ribosomal subunits is a highly dynamic process; pre-ribosomes undergo numerous structural rearrangements that establish the architecture present in mature complexes and serve as key checkpoints, ensuring the fidelity of ribosome assembly. Using in vivo crosslinking, we here identify the pre-ribosomal binding sites of three RNA helicases. Our data support roles for Has1 in triggering release of the U14 snoRNP, a critical event during early 40S maturation, and in driving assembly of domain I of pre-60S complexes. Binding of Mak5 to domain II of pre-60S complexes promotes recruitment of the ribosomal protein Rpl10, which is necessary for subunit joining and ribosome function. Spb4 binds to a molecular hinge at the base of ES27 facilitating binding of the export factor Arx1, thereby promoting pre-60S export competence. Our data provide important insights into the driving forces behind key structural remodelling events during ribosomal subunit assembly.

Deoxyribozymes are synthetic enzymes made of DNA that can catalyze the cleavage or formation of phosphodiester bonds and are useful tools for RNA biochemistry. Herein, we report new RNA-cleaving deoxyribozymes to interrogate the methylation status of target RNAs, thereby providing an alternative method for the biochemical validation of RNA methylation sites containing N 6 -methyladenosine, which is the most wide-spread and extensively investigated natural RNA modification. The developed deoxyribozymes are sensitive to the presence of N 6 -methyladenosine in RNA near the cleavage site. One class of these DNA enzymes shows faster cleavage of methylated RNA, while others are strongly inhibited by the modified nucleotide. The general applicability of the new deoxyribozymes is demonstrated for several examples of natural RNA sequences, including a lncRNA and a set of C/D box snoRNAs, which have been suggested to contain m 6 A as a regulatory element that influences RNA folding and protein binding., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Modifications in cellular RNAs have emerged as key regulators of all aspects of gene expression, including pre-mRNA splicing. During spliceosome assembly and function, the small nuclear RNAs (snRNAs) form numerous dynamic RNA-RNA and RNA-protein interactions, which are required for spliceosome assembly, correct positioning of the spliceosome on substrate pre-mRNAs and catalysis. The human snRNAs contain several base methylations as well as a myriad of pseudouridines and 2'-O-methylated nucleotides, which are largely introduced by small Cajal body-specific ribonucleoproteins (scaRNPs). Modified nucleotides typically cluster in functionally important regions of the snRNAs, suggesting that their presence could optimise the interactions of snRNAs with each other or with pre-mRNAs, or may affect the binding of spliceosomal proteins. snRNA modifications appear to play important roles in snRNP biogenesis and spliceosome assembly, and have also been proposed to influence the efficiency and fidelity of pre-mRNA splicing. Interestingly, alterations in the modification status of snRNAs have recently been observed in different cellular conditions, implying that some snRNA modifications are dynamic and raising the possibility that these modifications may fine-tune the spliceosome for particular functions. Here, we review the current knowledge on the snRNA modification machinery and discuss the timing, functions and dynamics of modifications in snRNAs.

N 6 -methyladenosine (m 6 A) modifications in RNAs play important roles in regulating many different aspects of gene expression. While m 6 As can have direct effects on the structure, maturation, or translation of mRNAs, such modifications can also influence the fate of RNAs via proteins termed "readers" that specifically recognize and bind modified nucleotides. Several YTH domain-containing proteins have been identified as m 6 A readers that regulate the splicing, translation, or stability of specific mRNAs. In contrast to the other YTH domain-containing proteins, YTHDC2 has several defined domains and here, we have analyzed the contribution of these domains to the RNA and protein interactions of YTHDC2. The YTH domain of YTHDC2 preferentially binds m 6 A-containing RNAs via a conserved hydrophobic pocket, whereas the ankyrin repeats mediate an RNA-independent interaction with the 5'-3' exoribonuclease XRN1. We show that the YTH and R3H domains contribute to the binding of YTHDC2 to cellular RNAs, and using crosslinking and analysis of cDNA (CRAC), we reveal that YTHDC2 interacts with the small ribosomal subunit in close proximity to the mRNA entry/exit sites. YTHDC2 was recently found to promote a "fast-track" expression program for specific mRNAs, and our data suggest that YTHDC2 accomplishes this by recruitment of the RNA degradation machinery to regulate the stability of m 6 A-containing mRNAs and by utilizing its distinct RNA-binding domains to bridge interactions between m 6 A-containing mRNAs and the ribosomes to facilitate their efficient translation., (© 2018 Kretschmer et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

RNA helicases are critical regulators at the nexus of multiple pathways of RNA metabolism, and in the complex cellular environment, tight spatial and temporal regulation of their activity is essential. Dedicated protein cofactors play key roles in recruiting helicases to specific substrates and modulating their catalytic activity. Alongside individual RNA helicase cofactors, networks of cofactors containing evolutionarily conserved domains such as the G-patch and MIF4G domains highlight the potential for cross-regulation of different aspects of gene expression. Structural analyses of RNA helicase-cofactor complexes now provide insight into the diverse mechanisms by which cofactors can elicit specific and coordinated regulation of RNA helicase action. Furthermore, post-translational modifications (PTMs) and long non-coding RNA (lncRNA) regulators have recently emerged as novel modes of RNA helicase regulation., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Mitochondrial protein synthesis is essential for the production of components of the oxidative phosphorylation system. RNA modifications in the mammalian mitochondrial translation apparatus play key roles in facilitating mitochondrial gene expression as they enable decoding of the non-conventional genetic code by a minimal set of tRNAs, and efficient and accurate protein synthesis by the mitoribosome. Intriguingly, recent transcriptome-wide analyses have also revealed modifications in mitochondrial mRNAs, suggesting that the concept of dynamic regulation of gene expression by the modified RNAs (the "epitranscriptome") extends to mitochondria. Furthermore, it has emerged that defects in RNA modification, arising from either mt-DNA mutations or mutations in nuclear-encoded mitochondrial modification enzymes, underlie multiple mitochondrial diseases. Concomitant advances in the identification of the mitochondrial RNA modification machinery and recent structural views of the mitochondrial translation apparatus now allow the molecular basis of such mitochondrial diseases to be understood on a mechanistic level.

N 6 -methyladenosine (m 6 A) is a highly dynamic RNA modification that has recently emerged as a key regulator of gene expression. While many m 6 A modifications are installed by the METTL3-METTL14 complex, others appear to be introduced independently, implying that additional human m 6 A methyltransferases remain to be identified. Using crosslinking and analysis of cDNA (CRAC), we reveal that the putative human m 6 A "writer" protein METTL16 binds to the U6 snRNA and other ncRNAs as well as numerous lncRNAs and pre-mRNAs. We demonstrate that METTL16 is responsible for N 6 -methylation of A43 of the U6 snRNA and identify the early U6 biogenesis factors La, LARP7 and the methylphosphate capping enzyme MEPCE as METTL16 interaction partners. Interestingly, A43 lies within an essential ACAGAGA box of U6 that base pairs with 5' splice sites of pre-mRNAs during splicing, suggesting that METTL16-mediated modification of this site plays an important role in splicing regulation. The identification of METTL16 as an active m 6 A methyltransferase in human cells expands our understanding of the mechanisms by which the m 6 A landscape is installed on cellular RNAs., (© 2017 The Authors.)

rRNAs are extensively modified during their transcription and subsequent maturation in the nucleolus, nucleus and cytoplasm. RNA modifications, which are installed either by snoRNA-guided or by stand-alone enzymes, generally stabilize the structure of the ribosome. However, they also cluster at functionally important sites of the ribosome, such as the peptidyltransferase center and the decoding site, where they facilitate efficient and accurate protein synthesis. The recent identification of sites of substoichiometric 2'-O-methylation and pseudouridylation has overturned the notion that all rRNA modifications are constitutively present on ribosomes, highlighting nucleotide modifications as an important source of ribosomal heterogeneity. While the mechanisms regulating partial modification and the functions of specialized ribosomes are largely unknown, changes in the rRNA modification pattern have been observed in response to environmental changes, during development, and in disease. This suggests that rRNA modifications may contribute to the translational control of gene expression.

In eukaryotes, the synthesis of ribosomal subunits, which involves the maturation of the ribosomal (r)RNAs and assembly of ribosomal proteins, requires the co-ordinated action of a plethora of ribosome biogenesis factors. Many of these cofactors remain to be characterized in human cells. Here, we demonstrate that the human G-patch protein NF-κB-repressing factor (NKRF) forms a pre-ribosomal subcomplex with the DEAH-box RNA helicase DHX15 and the 5΄-3΄ exonuclease XRN2. Using UV crosslinking and analysis of cDNA (CRAC), we reveal that NKRF binds to the transcribed spacer regions of the pre-rRNA transcript. Consistent with this, we find that depletion of NKRF, XRN2 or DHX15 impairs an early pre-rRNA cleavage step (A'). The catalytic activity of DHX15, which we demonstrate is stimulated by NKRF functioning as a cofactor, is required for efficient A' cleavage, suggesting that a structural remodelling event may facilitate processing at this site. In addition, we show that depletion of NKRF or XRN2 also leads to the accumulation of excised pre-rRNA spacer fragments and that NKRF is essential for recruitment of the exonuclease to nucleolar pre-ribosomal complexes. Our findings therefore reveal a novel pre-ribosomal subcomplex that plays distinct roles in the processing of pre-rRNAs and the turnover of excised spacer fragments., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Two proteins with PIN endonuclease domains, yUtp24(Fcf1)/hUTP24 and yUtp23/hUTP23 are essential for early pre-ribosomal (r)RNA cleavages at sites A0, A1/1 and A2/2a in yeast and humans. The yUtp24/hUTP24 PIN endonuclease is proposed to cleave at sites A1/1 and A2/2a, but the enzyme cleaving at site A0 is not known. Yeast yUtp23 contains a degenerate, non-essential PIN domain and functions together with the snR30 snoRNA, while human hUTP23 is associated with U17, the human snR30 counterpart. Using in vivo RNA-protein crosslinking and gel shift experiments, we reveal that yUtp23/hUTP23 makes direct contacts with expansion sequence 6 (ES6) in the 18S rRNA sequence and that yUtp23 interacts with the 3΄ half of the snR30 snoRNA. Protein-protein interaction studies further demonstrated that yeast yUtp23 and human hUTP23 directly interact with the H/ACA snoRNP protein yNhp2/hNHP2, the RNA helicase yRok1/hROK1(DDX52), the ribosome biogenesis factor yRrp7/hRRP7 and yUtp24/hUTP24. yUtp23/hUTP23 could therefore be central to the coordinated integration and release of ES6 binding factors and likely plays a pivotal role in remodeling this pre-rRNA region in both yeast and humans. Finally, studies using RNAi-rescue systems in human cells revealed that intact PIN domain and Zinc finger motifs in human hUTP23 are essential for 18S rRNA maturation., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The impact of RNA structures in coding sequences (CDS) within mRNAs is poorly understood. Here, we identify a novel and highly conserved mechanism of translational control involving RNA structures within coding sequences and the DEAD-box helicase Dhh1. Using yeast genetics and genome-wide ribosome profiling analyses, we show that this mechanism, initially derived from studies of the Brome Mosaic virus RNA genome, extends to yeast and human mRNAs highly enriched in membrane and secreted proteins. All Dhh1-dependent mRNAs, viral and cellular, share key common features. First, they contain long and highly structured CDSs, including a region located around nucleotide 70 after the translation initiation site; second, they are directly bound by Dhh1 with a specific binding distribution; and third, complementary experimental approaches suggest that they are activated by Dhh1 at the translation initiation step. Our results show that ribosome translocation is not the only unwinding force of CDS and uncover a novel layer of translational control that involves RNA helicases and RNA folding within CDS providing novel opportunities for regulation of membrane and secretome proteins., (© 2017 Jungfleisch et al.; Published by Cold Spring Harbor Laboratory Press.)

Several crosslinking methods have been developed to identify interacting RNAs for proteins of interest. Here, we describe variants of the UV crosslinking and analysis of cDNA (CRAC) method that allow target identification of RNA methyltransferases on a genome-wide scale. We present a detailed protocol for the application of CRAC in human cells that stably express the protein of interest fused to a tandem affinity tag. After the introduction of a covalent link between the protein and its target RNAs, protein-RNA complexes are purified and bound RNAs trimmed, ligated to adapters, reverse transcribed, and amplified. Sequences obtained from next-generation sequencing are then mapped onto the human genome allowing the identification of possible substrates. For some RNA methyltransferases, e.g., m 5 C MTases, their catalytic mechanism can be exploited for chemical crosslinking approaches instead of UV based crosslinking.

RNA methyltransferases (MTases) are responsible for co- and posttranscriptional methylation of nucleotides in a wide variety of RNA substrates. Examination of the target specificity, catalytic activity, and function of these enzymes requires in vitro methylation assays. Here, we provide a detailed protocol for the methylation of in vitro transcripts, synthetic RNAs, and total cellular RNA using recombinant RNA methyltransferases and S-adenosylmethionine (SAM) as a methyl group donor. We describe how this method can be coupled to fluorographic detection of RNA methylation if 3 H-labeled SAM is used, and discuss alternative chromatography-based methods for the detection of methylated nucleotides, focusing on reversed-phase high-performance liquid chromatography (RP-HPLC). In both cases, mutagenesis of the methyltransferase or the RNA substrate can be employed to elucidate the catalytic mechanisms and target specificity of the enzymes. Together these approaches provide valuable insight into the action of RNA methyltransferases on the molecular level and serve as a basis for further functional characterization of RNA methyltransferases in vivo.

Bowen-Conradi syndrome (BCS) is a severe genetic disorder that is characterised by various developmental abnormalities, bone marrow failure and early infant death. This disease is caused by a single mutation leading to the aspartate 86 to glycine (D86G) exchange in the essential nucleolar RNA methyltransferase EMG1. EMG1 is required for the synthesis of the small ribosomal subunit and is involved in modification of the 18S ribosomal RNA. Here, we identify the pre-ribosomal factors NOP14, NOC4L and UTP14A as members of a nucleolar subcomplex that contains EMG1 and is required for its recruitment to nucleoli. The BCS mutation in EMG1 leads to reduced nucleolar localisation, accumulation of EMG1D86G in nuclear foci and its proteasome-dependent degradation. We further show that EMG1 can be imported into the nucleus by the importins (Imp) Impα/β or Impβ/7. Interestingly, in addition to its role in nuclear import, binding of the Impβ/7 heterodimer can prevent unspecific aggregation of both EMG1 and EMG1D86G on RNAs in vitro, indicating that the importins act as chaperones by binding to basic regions of the RNA methyltransferase. Our findings further indicate that in BCS, nuclear disassembly of the import complex and release of EMG1D86G lead to its nuclear aggregation and degradation, resulting in the reduced nucleolar recruitment of the RNA methyltransferase and defects in the biogenesis of the small ribosomal subunit., (© The Author 2016. Published by Oxford University Press.)

Mitochondrial gene expression uses a non-universal genetic code in mammals. Besides reading the conventional AUG codon, mitochondrial (mt-)tRNA M et mediates incorporation of methionine on AUA and AUU codons during translation initiation and on AUA codons during elongation. We show that the RNA methyltransferase NSUN3 localises to mitochondria and interacts with mt-tRNA M et to methylate cytosine 34 (C34) at the wobble position. NSUN3 specifically recognises the anticodon stem loop (ASL) of the tRNA, explaining why a mutation that compromises ASL basepairing leads to disease. We further identify ALKBH1/ABH1 as the dioxygenase responsible for oxidising m 5 C34 of mt-tRNA M et to generate an f 5 C34 modification. In vitro codon recognition studies with mitochondrial translation factors reveal preferential utilisation of m 5 C34 mt-tRNA Met in initiation. Depletion of either NSUN3 or ABH1 strongly affects mitochondrial translation in human cells, implying that modifications generated by both enzymes are necessary for mt-tRNA M et function. Together, our data reveal how modifications in mt-tRNA M et are generated by the sequential action of NSUN3 and ABH1, allowing the single mitochondrial tRNA M et to recognise the different codons encoding methionine., (© 2016 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

RNAs and ribonucleoprotein complexes (RNPs) play key roles in mediating and regulating gene expression. In eukaryotes, most RNAs are transcribed, processed and assembled with proteins in the nucleus and then either function in the cytoplasm or also undergo a cytoplasmic phase in their biogenesis. This compartmentalization ensures that sequential steps in gene expression and RNP production are performed in the correct order and it allows important quality control mechanisms that prevent the involvement of aberrant RNAs/RNPs in these cellular pathways. The selective exchange of RNAs/RNPs between the nucleus and cytoplasm is enabled by nuclear pore complexes, which function as gateways between these compartments. RNA/RNP transport is facilitated by a range of nuclear transport receptors and adaptors, which are specifically recruited to their cargos and mediate interactions with nucleoporins to allow directional translocation through nuclear pore complexes. While some transport factors are only responsible for the export/import of a certain class of RNA/RNP, others are multifunctional and, in the case of large RNPs, several export factors appear to work together to bring about export. Recent structural studies have revealed aspects of the mechanisms employed by transport receptors to enable specific cargo recognition, and genome-wide approaches have provided the first insights into the diverse composition of pre-mRNPs during export. Furthermore, the regulation of RNA/RNP export is emerging as an important means to modulate gene expression under stress conditions and in disease., (Copyright © 2015. Published by Elsevier Ltd.)

A rapidly increasing number of RNA helicases are implicated in several distinct cellular processes, however, the modes of regulation of multifunctional RNA helicases and their recruitment to different target complexes have remained unknown. Here, we show that the distribution of the multifunctional DEAH-box RNA helicase Prp43 between its diverse cellular functions can be regulated by the interplay of its G-patch protein cofactors. We identify the orphan G-patch protein Cmg1 (YLR271W) as a novel cofactor of Prp43 and show that it stimulates the RNA binding and ATPase activity of the helicase. Interestingly, Cmg1 localizes to the cytoplasm and to the intermembrane space of mitochondria and its overexpression promotes apoptosis. Furthermore, our data reveal that different G-patch protein cofactors compete for interaction with Prp43. Changes in the expression levels of Prp43-interacting G-patch proteins modulate the cellular localization of Prp43 and G-patch protein overexpression causes accumulation of the helicase in the cytoplasm or nucleoplasm. Overexpression of several G-patch proteins also leads to defects in ribosome biogenesis that are consistent with withdrawal of the helicase from this pathway. Together, these findings suggest that the availability of cofactors and the sequestering of the helicase are means to regulate the activity of multifunctional RNA helicases and their distribution between different cellular processes.

Translation fidelity and efficiency require multiple ribosomal (r)RNA modifications that are mostly mediated by small nucleolar (sno)RNPs during ribosome production. Overlapping basepairing of snoRNAs with pre-rRNAs often necessitates sequential and efficient association and dissociation of the snoRNPs, however, how such hierarchy is established has remained unknown so far. Here, we identify several late-acting snoRNAs that bind pre-40S particles in human cells and show that their association and function in pre-40S complexes is regulated by the RNA helicase DDX21. We map DDX21 crosslinking sites on pre-rRNAs and show their overlap with the basepairing sites of the affected snoRNAs. While DDX21 activity is required for recruitment of the late-acting snoRNAs SNORD56 and SNORD68, earlier snoRNAs are not affected by DDX21 depletion. Together, these observations provide an understanding of the timing and ordered hierarchy of snoRNP action in pre-40S maturation and reveal a novel mode of regulation of snoRNP function by an RNA helicase in human cells., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Ribosome biogenesis in yeast requires 75 small nucleolar RNAs (snoRNAs) and a myriad of cofactors for processing, modification, and folding of the ribosomal RNAs (rRNAs). For the 19 RNA helicases implicated in ribosome synthesis, their sites of action and molecular functions have largely remained unknown. Here, we have used UV cross-linking and analysis of cDNA (CRAC) to reveal the pre-rRNA binding sites of the RNA helicase Rok1, which is involved in early small subunit biogenesis. Several contact sites were identified in the 18S rRNA sequence, which interestingly all cluster in the "foot" region of the small ribosomal subunit. These include a major binding site in the eukaryotic expansion segment ES6, where Rok1 is required for release of the snR30 snoRNA. Rok1 directly contacts snR30 and other snoRNAs required for pre-rRNA processing. Using cross-linking, ligation and sequencing of hybrids (CLASH) we identified several novel pre-rRNA base-pairing sites for the snoRNAs snR30, snR10, U3, and U14, which cluster in the expansion segments of the 18S rRNA. Our data suggest that these snoRNAs bridge interactions between the expansion segments, thereby forming an extensive interaction network that likely promotes pre-rRNA maturation and folding in early pre-ribosomal complexes and establishes long-range rRNA interactions during ribosome synthesis., (© 2014 Martin et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

During eukaryotic ribosome biogenesis, three of the mature ribosomal (r)RNAs are released from a single precursor transcript (pre-rRNA) by an ordered series of endonucleolytic cleavages and exonucleolytic processing steps. Production of the 18S rRNA requires the removal of the 5' external transcribed spacer (5'ETS) by endonucleolytic cleavages at sites A0 and A1/site 1. In metazoans, an additional cleavage in the 5'ETS, at site A', upstream of A0, has also been reported. Here, we have investigated how A' processing is coordinated with assembly of the early preribosomal complex. We find that only the tUTP (UTP-A) complex is critical for A' cleavage, while components of the bUTP (UTP-B) and U3 snoRNP are important, but not essential, for efficient processing at this site. All other factors involved in the early stages of 18S rRNA processing that were tested here function downstream from this processing step. Interestingly, we show that the RNA surveillance factors XRN2 and MTR4 are also involved in A' cleavage in humans. A' cleavage is largely bypassed when XRN2 is depleted, and we also discover that A' cleavage is not always the initial processing event in all cell types. Together, our data suggest that A' cleavage is not a prerequisite for downstream pre-rRNA processing steps and may, in fact, represent a quality control step for initial pre-rRNA transcripts. Furthermore, we show that components of the RNA surveillance machinery, including the exosome and TRAMP complexes, also play key roles in the recycling of excised spacer fragments and degradation of aberrant pre-rRNAs in human cells.

Several proto-oncogenes and tumor suppressors regulate the production of ribosomes. Ribosome biogenesis is a major consumer of cellular energy, and defects result in p53 activation via repression of mouse double minute 2 (MDM2) homolog by the ribosomal proteins RPL5 and RPL11. Here, we report that RPL5 and RPL11 regulate p53 from the context of a ribosomal subcomplex, the 5S ribonucleoprotein particle (RNP). We provide evidence that the third component of this complex, the 5S rRNA, is critical for p53 regulation. In addition, we show that the 5S RNP is essential for the activation of p53 by p14(ARF), a protein that is activated by oncogene overexpression. Our data show that the abundance of the 5S RNP, and therefore p53 levels, is determined by factors regulating 5S complex formation and ribosome integration, including the tumor suppressor PICT1. The 5S RNP therefore emerges as the critical coordinator of signaling pathways that couple cell proliferation with ribosome production., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Human ribosome production is up-regulated during tumorogenesis and is defective in many genetic diseases (ribosomopathies). We have undertaken a detailed analysis of human precursor ribosomal RNA (pre-rRNA) processing because surprisingly little is known about this important pathway. Processing in internal transcribed spacer 1 (ITS1) is a key step that separates the rRNA components of the large and small ribosomal subunits. We report that this was initiated by endonuclease cleavage, which required large subunit biogenesis factors. This was followed by 3' to 5' exonucleolytic processing by RRP6 and the exosome, an enzyme complex not previously linked to ITS1 removal. In contrast, RNA interference-mediated knockdown of the endoribonuclease MRP did not result in a clear defect in ITS1 processing. Despite the apparently high evolutionary conservation of the pre-rRNA processing pathway and ribosome synthesis factors, each of these features of human ITS1 processing is distinct from those in budding yeast. These results also provide significant insight into the links between ribosomopathies and ribosome production in human cells.

Most RNAs in eukaryotic cells are produced as precursors that undergo processing at the 3' and/or 5' end to generate the mature transcript. In addition, many transcripts are degraded not only as part of normal recycling, but also when recognized as aberrant by the RNA surveillance machinery. The exosome, a conserved multiprotein complex containing two nucleases, is involved in both the 3' processing and the turnover of many RNAs in the cell. A series of factors, including the TRAMP (Trf4-Air2-Mtr4 polyadenylation) complex, Mpp6 and Rrp47, help to define the targets to be processed and/or degraded and assist in exosome function. The majority of the data on the exosome and RNA maturation/decay have been derived from work performed in the yeast Saccharomyces cerevisiae. In the present paper, we provide an overview of the exosome and its role in RNA processing/degradation and discuss important new insights into exosome composition and function in human cells.

2'-O-methylation of eukaryotic ribosomal RNA (r)RNA, essential for ribosome function, is catalysed by box C/D small nucleolar (sno)RNPs. The RNA components of these complexes (snoRNAs) contain one or two guide sequences, which, through base-pairing, select the rRNA modification site. Adjacent to the guide sequences are protein-binding sites (the C/D or C'/D' motifs). Analysis of >2000 yeast box C/D snoRNAs identified additional conserved sequences in many snoRNAs that are complementary to regions adjacent to the rRNA methylation site. This 'extra base-pairing' was also found in many human box C/D snoRNAs and can stimulate methylation by up to five-fold. Sequence analysis, combined with RNA-protein crosslinking in Saccharomyces cerevisiae, identified highly divergent box C'/D' motifs that are bound by snoRNP proteins. In vivo rRNA methylation assays showed these to be active. Our data suggest roles for non-catalytic subunits (Nop56 and Nop58) in rRNA binding and support an asymmetric model for box C/D snoRNP organization. The study provides novel insights into the extent of the snoRNA-rRNA interactions required for efficient methylation and the structural organization of the snoRNPs.

Our aim was to assess the intra- and inter-observer reliability in the establishment of the anterior pelvic plane used in imageless computer-assisted navigation. From this we determined the subsequent effects on version and inclination of the acetabular component. A cadaver model was developed with a specifically-designed rod which held the component tracker at a fixed orientation to the pelvis, leaving the anterior pelvic plane as the only variable. Eight surgeons determined the anterior pelvic plane by palpating and registering the bony landmarks as reference points. The exact anterior pelvic plane was then established by using anatomically-placed bone screws as reference points. The difference between the surgeons was found to be highly significant (p < 0.001). The variation was significantly larger for anteversion (sd 9.6 degrees ) than for inclination (sd 6.3 degrees ). The present method for registering pelvic landmarks shows significant inaccuracy, which highlights the need for improved methods of registration before this technique is considered to be safe.

We recently identified the immunoglobulin-CAM CD155/PVR (the poliovirus receptor) as a regulator of cancer invasiveness and glioma migration, but the mechanism through which CD155/PVR controls these processes is unknown. Here, we show that expression of CD155/PVR in rat glioma cells that normally lack this protein enhances their dispersal both in vitro and on primary brain tissue. CD155/PVR expression also reduced substrate adhesion, cell spreading, focal adhesion density, and the number of actin stress fibers in a substrate-dependent manner. Furthermore, we found that expression of CD155/PVR increased Src/focal adhesion kinase signaling in a substrate-dependent manner, enhancing the adhesion-induced activation of paxillin and p130Cas in cells adhering to vitronectin. Conversely, depletion of endogenous CD155/PVR from human glioma cells inhibited their migration, increased cell spreading, and down-regulated the same signaling pathway. These findings implicate CD155/PVR as a regulator of adhesion signaling and suggest a pathway through which glioma and other cancer cells may acquire a dispersive phenotype.

Apoptotic evasion is a hallmark of cancer and its resistance to chemotherapeutic drugs. Identification of cellular proteins that mediate apoptotic programs is a critical step toward the development of therapeutics aimed at overcoming apoptosis resistance. We developed an innovative high-throughput screen to identify proteins that modulate Fas ligand-mediated apoptosis using fluorophore-assisted light inactivation (HTS-FALIpop). The FALI protein knockdown strategy was coupled to a caspase activity assay with the ability to detect both proapoptotic and antiapoptotic surface molecules expressed by HT-1080 human fibrosarcoma cells. FALI of the Fas receptor (Fas/CD95) using a fluorescein-conjugated anti-Fas antibody abrogated Fas ligand-mediated caspase activation. Ninety-six single-chain variable fragment antibodies (scFv), selected for binding to the surface of HT-1080 cells, were screened by HTS-FALIpop. Three of the scFvs caused decreases in caspase induction after FALI of their protein targets. One of the targets of these positive scFvs was identified as CD44 and was validated by performing FALI using a CD44-specific monoclonal antibody, which resulted in similar protection from Fas apoptosis. CD44-targeted FALI was antiapoptotic in multiple human cancer cell lines, including both Fas signaling type I and II cells, and was also protective against other ligands of the tumor necrosis factor death receptor family. FALI of CD44 inhibited formation and activation of the death-inducing signaling complex, suggesting that CD44 regulates Fas at the cell surface. This mechanism of death receptor regulation represents a novel means of apoptosis modulation that could be exploited by pharmacologic agents.

Background and Purpose: The present study was performed to determine the role of alpha4 (CD49d), a member of the integrin family of adhesion molecules, in ischemic brain pathology., Methods: Male spontaneously hypertensive rats (SHR) or Sprague-Dawley rats underwent 60-minute middle cerebral artery occlusion (MCAO) followed by 23-hour reperfusion. Animals were injected intravenously with 2.5 mg/kg anti-rat alpha4 antibody (TA-2) or isotype control antibody (anti-human LFA-3 IgG(1), 1E6) 24 hours before MCAO. Infarct volume was quantified by staining of fresh tissue with tetrazolium chloride and myeloperoxidase activity measured in SHR tissue homogenates 24 hours after MCAO. In SHR, mean arterial blood pressure was recorded before and after MCAO in animals treated with TA-2 and 1E6. Fluorescence-activated cell sorting analysis was performed on peripheral blood leukocytes before and after MCAO., Results: TA-2 treatment significantly reduced total infarct volume by 57.7% in normotensive rats (1E6, 84.2+/-11.5 mm(3), n=17; TA-2, 35.7+/-5.9 mm(3), n=16) and 35.5% in hypertensive rats (1E6, 146.6+/-15.5 mm(3), n=15; TA-2, 94.4+/-25.8 mm(3), n=11). In both strains, TA-2 treatment significantly reduced body weight loss and attenuated the hyperthermic response to MCAO. In SHR, treatment with TA-2 significantly reduced brain myeloperoxidase activity. Resting mean arterial blood pressure was unaffected by treatment. Leukocyte counts were elevated in TA-2-treated rats. Fluorescence-activated cell sorting analysis demonstrated the ability of TA-2 to bind to CD3+, CD4+, CD8+, and CD11b+ cells in both naive animals and after MCAO., Conclusions: These data demonstrate that inhibition of alpha4 integrin can protect the brain against ischemic brain injury and implicate endogenous alpha4 integrin in the pathogenesis of acute brain injury. The mechanism by which alpha4 integrin inhibition offers cerebroprotection is independent of blood pressure modulation and is likely due to inhibition of leukocyte function.

Twelve patients were involved in a 3 month stimulation induced cycling programme at the Royal Perth Rehabilitation Hospital. A number of the patients were less than 1 year post injury, all except one had an incomplete injury, and most were receiving physiotherapy. All patients who completed the programme increased their time of cycling and, in all but one case, the exercise load, indicative of a local training effect. Significant improvements were found in voluntary isometric strength, stimulated isometric strength and stimulated isometric endurance of the quadriceps, muscle grading of the quadriceps and biceps femoris and the cross-sectional areas of the quadriceps and total thigh muscle. No change was found in voluntary isokinetic strength of the quadriceps. All patients with incomplete injuries reported improvements in the activities of daily living (ADL) after the programme. Bone mineral density (BMD) was examined in two patients, one less than 1 year post injury, and one greater than 4 years post injury. The programme of cycling did not restore BMD in the latter patient. However, while the former patient still displayed a reduced BMD after the programme, it is unknown whether this loss of bone was retarded. This needs further investigation. This study demonstrates the effectiveness of a combined physiotherapy/cycling programme in the rehabilitation of people with spinal injuries. To be successful this type of programme has to be incorporated into the rehabilitation process, as has been done at the Sir George Bedbrook Spinal Unit.

A low cost clinical exercise system was developed for the spinal cord injured, based on a bicycle ergometer and electrical stimulation. A pilot project was conducted, using the system, to examine the effects of stimulation induced cycling in long term paraplegics. The project comprised 2 phases of exercise, a strengthening phase involving a 12 week programme of electrical stimulation to the quadriceps and hamstrings and a 12 week cycling phase. Physiological, morphological and biochemical parameters were measured for each subject, at the beginning of the programme and following each phase. Results showed that a programme of stimulation induced lower limb exercise increased the exercise tolerance of all patients, as determined by a progressive increase in exercise time, cycling rate and exercise load. The enhanced exercise tolerance was a result of increases in local muscle strength and endurance. Increases in thigh muscle area and joint range of motion were recorded and all incomplete subjects reported an improvement in functional capabilities and general wellbeing.

The distribution of phosphorylated and non-phosphorylated neurofilament epitopes was determined immunocytochemically in adjacent 2 microns-thick sections of sciatic nerve, ventral root and spinal cord. Staining was scored as either intense, moderate or absent and the proportion of labeled axons was calculated for each category. Nearly all sciatic nerve and ventral root axons were immunoreactive with both antibodies against phosphorylated and non-phosphorylated neurofilaments and there were no significant differences in the number of intensely- or moderately-labeled axons. Within the spinal cord however, while the majority of large caliber axons was stained with both antibodies, there was a significant number of small caliber axons which stained only with antibodies against phosphorylated neurofilaments. These results show that phosphorylated and non-phosphorylated neurofilaments are extensively codistributed in CNS and PNS axons, and that in the CNS, staining intensity for non-phosphorylated epitopes is less in the smaller axons.

Retinae and optic nerve sections from adult hamsters were reacted with antibodies against phosphorylated (P) or non-phosphorylated (NP) neurofilament proteins. NP epitopes were observed within ganglion cell bodies and extended into the proximal portion of the optic nerve. P epitopes became prominent within axons as they approached the optic disc and remained throughout the optic nerve. This spatial distribution appears similar to the pattern of myelination within this axonal population.