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In this review, I describe our scientific journey to unearth the impact of RNA metabolism in cancer using the eukaryotic translation initiation factor eIF4E as an exemplar. This model allowed us to discover new structural, biochemical, and molecular features of RNA processing, and to reveal their substantial impact on cell physiology. This led us to develop proof-of-principle strategies to target these pathways in cancer patients leading to clinical benefit. I discuss the important role that the unexpected plays in research and the necessity of embracing the data even when it clashes with dogma. I also touch on the importance of equity, diversity, and inclusion to the success of the scientific enterprise. Key words: eIF4E, RNA processing, cancer, acute myeloid leukemia Dans cette revue, je decris notre cheminement scientifique pour decouvrir l'impact du metabolisme de TARN dans le cancer en prenant comme modele le facteur d'initiation de la traduction eucaryote eIF4E. Ce modele nous a permis de decouvrir de nouvelles caracteristiques structurelles, biochimiques et moleculaires de la production de l'ARN et de reveler leur impact substantiel sur la physiologie cellulaire. Cela nous a amenes a developper des strategies pour cibler ces voies chez les patients cancereux conduisant a un benefice clinique. Ici, je discute du role important que joue l'imprevu dans la recherche et de la necessite d'embrasser les donnees meme lorsqu'elles se heurtent aux dogmes. J'aborde egalement l'importance de l'equite, de la diversite et de l'inclusion pour le succes de l'entreprise scientifique. Mots-cles : eIF4E, traitement de l'ARN, cancer, leucemie myeloide aigue, A bit of backstory The central dogma of molecular biology positions mRNA as a simple intermediary in a process whereby DNA instructs the cells to generate protein actors to carry [...]

Approximately 10% of monogenic diseases are caused by nonsense point mutations that generate premature termination codons (PTCs), resulting in a truncated protein and nonsense-mediated decay of the mutant mRNAs. Here, we demonstrate a mini-dCas13X-mediated RNA adenine base editing (mxABE) strategy to treat nonsense mutation-related monogenic diseases via A-to-G editing in a genetically humanized mouse model of Duchenne muscular dystrophy (DMD). Initially, we identified a nonsense point mutation (c.4174C>T, p.Gln1392*) in the DMD gene of a patient and validated its pathogenicity in humanized mice. In this model, mxABE packaged in a single adeno-associated virus (AAV) reached A-to-G editing rates up to 84% in vivo, at least 20-fold greater than rates reported in previous studies using other RNA editing modalities. Furthermore, mxABE restored robust expression of dystrophin protein to over 50% of WT levels by enabling PTC read-through in multiple muscle tissues. Importantly, systemic delivery of mxABE by AAV also rescued dystrophin expression to averages of 37%, 6%, and 54% of WT levels in the diaphragm, tibialis anterior, and heart muscle, respectively, as well as rescued muscle function. Our data strongly suggest that mxABE-based strategies may be a viable new treatment modality for DMD and other monogenic diseases., Introduction The majority of monogenic diseases are caused by point mutations (1), among which approximately 50% can be reversed by A-to-G conversion with adenine base editors (ABEs). Specifically, A-to-G conversion [...]

Author(s): Meng How Tan (corresponding author) [1, 2, 3]; Qin Li [1]; Raghuvaran Shanmugam [2, 3]; Robert Piskol [1]; Jennefer Kohler [1]; Amy N. Young [1]; Kaiwen Ivy Liu [3]; [...]

DNase I hypersensitive sites (DHSs) provide important information on the presence of transcriptional regulatory elements and the state of chromatin in mammalian cells (1-3). Conventional DNase sequencing (DNase-seq) for genome-wide DHSs profiling is limited by the requirement of millions of cells (4,5). Here we report an ultrasensitive strategy, called single-cell DNase sequencing (scDNase-seq) for detection of genome-wide DHSs in single cells. We show that DHS patterns at the single-cell level are highly reproducible among individual cells. Among different single cells, highly expressed gene promoters and enhancers associated with multiple active histone modifications display constitutive DHS whereas chromatin regions with fewer histone modifications exhibit high variation of DHS. Furthermore, the single-cell DHSs predict enhancers that regulate cell-specific gene expression programs and the cell-to-cell variations of DHS are predictive of gene expression. Finally, we apply scDNase-seq to pools of tumour cells and pools of normal cells, dissected from formalin-fixed paraffin-embedded tissue slides from patients with thyroid cancer, and detect thousands of tumour-specific DHSs. Many of these DHSs are associated with promoters and enhancers critically involved in cancer development. Analysis of the DHS sequences uncovers one mutation (chr18: 52417839G > C) in the tumour cells of a patient with follicular thyroid carcinoma, which affects the binding of the tumour suppressor protein p53 and correlates with decreased expression of its target gene TXNL1. In conclusion, scDNase-seq can reliably detect DHSs in single cells, greatly extending the range of applications of DHS analysis both for basic and for translational research, and may provide critical information for personalized medicine., We developed a circular carrier DNA-mediated sequencing method, called scDNase-seq, to analyse genome-wide DHSs in a few cells or even single cells (Fig. 1a). Application of scDNase-seq to NIH3T3 cells [...]

The single-stranded nature of RNAs synthesized in the cell gives them great scope to form different structures, but current methods to measure RNA structure in vivo are limited; now, a new methodology allows researchers to examine all four nucleotides in mouse embryonic stem cells. Probing native RNA structure The single-stranded nature of cellular RNAs allows them flexibility to adopt different secondary structures that can affect their function. However, current methods of measuring RNA structure in vivo are limited. Two papers published in this week's issue of Nature present new techniques to address this gap. Howard Chang and colleagues have exploited a click methodology that enables the first global view of RNA secondary structures in living cells for all four bases. While some structures are stable and seem to be programmed by sequence, others are dynamic, reflecting the binding of proteins or modification of the bases. This method may allow RNA to be analysed in vivo from a structural genomics perspective. In the second study, Jernej Ule and colleagues have developed a method, hiCLIP, to specifically measure RNA structures bound by proteins. Various features are observed, such as a preference for intramolecular interactions and an under-representation of structures in coding regions. The results confirm that RNA structure is able to regulate gene expression. While the functional significance is not known, it is notable that SNPs are not present at the expected frequency in coding regions. Visualizing the physical basis for molecular behaviour inside living cells is a great challenge for biology. RNAs are central to biological regulation, and the ability of RNA to adopt specific structures intimately controls every step of the gene expression program.sup.1. However, our understanding of physiological RNA structures is limited; current in vivo RNA structure profiles include only two of the four nucleotides that make up RNA.sup.2,3. Here we present a novel biochemical approach, in vivo click selective 2'-hydroxyl acylation and profiling experiment (icSHAPE), which enables the first global view, to our knowledge, of RNA secondary structures in living cells for all four bases. icSHAPE of the mouse embryonic stem cell transcriptome versus purified RNA folded in vitro shows that the structural dynamics of RNA in the cellular environment distinguish different classes of RNAs and regulatory elements. Structural signatures at translational start sites and ribosome pause sites are conserved from in vitro conditions, suggesting that these RNA elements are programmed by sequence. In contrast, focal structural rearrangements in vivo reveal precise interfaces of RNA with RNA-binding proteins or RNA-modification sites that are consistent with atomic-resolution structural data. Such dynamic structural footprints enable accurate prediction of RNA-protein interactions and N.sup.6-methyladenosine (m.sup.6A) modification genome wide. These results open the door for structural genomics of RNA in living cells and reveal key physiological structures controlling gene expression., Author(s): Robert C. Spitale [sup.1] , Ryan A. Flynn [sup.1] , Qiangfeng Cliff Zhang [sup.1] , Pete Crisalli [sup.2] , Byron Lee [sup.1] , Jong-Wha Jung [sup.2] , Hannes Y. [...]

Antigenic variation of the Plasmodium falciparum multicopy var gene family enables parasite evasion of immune destruction by host antibodies (1,2). Expression of a particular var subgroup, termed upsA, is linked to the obstruction of blood vessels in the brain and to the pathogenesis of human cerebral malaria (3-6). The mechanism determining upsA activation remains unknown. Here we show that an entirely new type of gene silencing mechanism involving an exonuclease-mediated degradation of nascent RNA controls the silencing of genes linked to severe malaria. We identify a novel chromatin-associated exoribonuclease, termed PfRNase II, that controls the silencing of upsA var genes by marking their transcription start site and intron-promoter regions leading to short-lived cryptic RNA. Parasites carrying a deficient PfRNase II gene produce full-length upsA var transcripts and intron-derived antisense long non-coding RNA. The presence of stable upsA var transcripts overcomes monoallelic expression, resulting in the simultaneous expression of both upsA and upsC type PfEMP1 proteins on the surface of individual infected red blood cells. In addition, we observe an inverse relationship between transcript levels of PfRNase II and ups. A-type var genes in parasites from severe malaria patients, implying a crucial role of PfRNase II in severe malaria. Our results uncover a previously unknown type of posttranscriptional gene silencing mechanism in malaria parasites with repercussions for other organisms. Additionally, the identification of RNase II as a parasite protein controlling the expression of virulence genes involved in pathogenesis in patients with severe malaria may provide new strategies for reducing malaria mortality., Beyond histone modifying enzymes (7-10), additional post-transcriptional mechanisms may control antigenic variation of the P. falciparum multicopy var gene family. RNA processing and degradation in P. falciparum erythrocytic-stage parasites has [...]

Programmed -1 ribosomal frameshift (-1PRF) signals redirect translating ribosomes to slip back one base on messenger RNAs. Although well characterized in viruses, how these elements may regulate cellular gene expression is not understood. Here we describe a -1 PRF signal in the human mRNA encoding CCR5, the HIV-1 co- receptor. CCR5 mRNA-mediated -1 PRF is directed by an mRNA pseudoknot, and is stimulated by at least two microRNAs. Mapping the mRNA-miRNA interaction suggests that formation of a triplex RNA structure stimulates -1 PRF. A -1 PRF event on the CCR5 mRNA directs translating ribosomes to a premature termination codon, destabilizing it through the nonsense-mediated mRNA decay pathway. At least one additional mRNA decay pathway is also involved. Functional -1 PRF signals that seem to be regulated by miRNAs are also demonstrated in mRNAs encoding six other cytokine receptors, suggesting a novel mode through which immune responses may be fine-tuned in mammalian cells., Viral programmed ribosomal frameshift events typically produce carboxy-terminally extended fusion proteins. However, computational analyses predict that >95% of -1 PRF events on cellular mRNAs direct ribosomes to premature termination codons [...]

Only a very small fraction of long noncoding RNAs (lncRNAs) are well characterized. The evolutionary history of lncRNAs can provide insights into their functionality, but the absence of lncRNA annotations in non-model organisms has precluded comparative analyses. Here we present a large-scale evolutionary study of lncRNA repertoires and expression patterns, in 11 tetrapod species. We identify approximately 11, 000 primate-specific lncRNAs and 2, 500 highly conserved lncRNAs, including approximately 400 genes that are likely to have originated more than 300 million years ago. We find that lncRNAs, in particular ancient ones, are in general actively regulated and may function predominantly in embryonic development. Most lncRNAs evolve rapidly in terms of sequence and expression levels, but tissue specificities are often conserved. We compared expression patterns of homologous lncRNA and protein-coding families across tetrapods to reconstruct an evolutionarily conserved co- expression network. This network suggests potential functions for lncRNAs in fundamental processes such as spermatogenesis and synaptic transmission, but also in more specific mechanisms such as placenta development through microRNA production., Evolutionary analyses of protein-coding gene sequences (1) and expression patterns (2) have provided important insights into the genetic basis of lineage-specific phenotypes and into individual gene functions. For lncRNAs, such [...]

Nucleic-acid-binding proteins are generally viewed as either specific or nonspecific, depending on characteristics of their binding sites in DNA or RNA (1,2). Most studies have focused on specific proteins, which identify cognate sites by binding with highest affinities to regions with defined signatures in sequence, structure or both (1-4). Proteins that bind to sites devoid of defined sequence or structure signatures are considered nonspecific (1,2,5). Substrate binding by these proteins is poorly understood, and it is not known to what extent seemingly nonspecific proteins discriminate between different binding sites, aside from those sequestered by nucleic acid structures (6). Here we systematically examine substrate binding by the apparently nonspecific RNA-binding protein C5, and find clear discrimination between different binding site variants. C5 is the protein subunit of the transfer RNA processing ribonucleoprotein enzyme RNase P from Escherichia coli. The protein binds 5' leaders of precursor tRNAs at a site without sequence or structure signatures. We measure functional binding of C5 to all possible sequence variants in its substrate binding site, using a high-throughput sequencing kinetics approach (HITS-KIN) that simultaneously follows processing of thousands of RNA species. C5 binds different substrate variants with affinities varying by orders of magnitude. The distribution of functional affinities of C5 for all substrate variants resembles affinity distributions of highly specific nucleic acid binding proteins. Unlike these specific proteins, C5 does not bind its physiological RNA targets with the highest affinity, but with affinities near the median of the distribution, a region that is not associated with a sequence signature. We delineate defined rules governing substrate recognition by C5, which reveal specificity that is hidden in cellular substrates for RNase P. Our findings suggest that apparently nonspecific and specific RNA-binding modes may not differ fundamentally, but represent distinct parts of common affinity distributions., (9) The term 'nonspecific' is widely used to describe proteins that bind DNA or RNA substrates at sites without apparent sequence or structure signatures (1,2,5). Although nonspecific proteins are numerous [...]

The 7-methylguanosine cap structure at the 5' end of eukaryotic messenger RNAs is a critical determinant of their stability and translational efficiency (1-3). It is generally believed that 5'-end capping is a constitutive process that occurs during mRNA maturation and lacks the need for a quality-control mechanism to ensure its fidelity. We recently reported that the yeast Rail protein has pyrophosphohydrolase activity towards mRNAs lacking a 5'-end cap (4). Here we show that, in vitro as well as in yeast cells, Rail possesses a novel decapping endonuclease activity that can also remove the entire cap structure dinucleotide from an mRNA. This activity is targeted preferentially towards mRNAs with unmethylated caps in contrast to the canonical decapping enzyme, Dcp2, which targets mRNAs with a methylated cap. Capped but unmethylated mRNAs generated in yeast cells with a defect in the methyltransferase gene are more stable in a rail-gene-disrupted background. Moreover, railΔ yeast cells with wild-type capping enzymes show significant accumulation of mRNAs with 5' -end capping defects under nutritional stress conditions of glucose starvation or amino acid starvation. These findings provide evidence that 5' -end capping is not a constitutive process that necessarily always proceeds to completion and demonstrates that Rail has an essential role in clearing mRNAs with aberrant 5'-end caps. We propose that Rail is involved in an as yet uncharacterized quality control process that ensures mRNA 5' -end integrity by an aberrant-cap-mediated mRNA decay mechanism., The stability and translational efficiency of eukaryotic mRNAs are both influenced by the 5'-end cap (1-3). The cap is co-transcriptionally added and consists of a guanine nucleoside methylated at the [...]

Protein modification by conjugation of small ubiquitin-related modifier (SUMO) is involved in diverse biological functions, such as transcription regulation, subcellular partitioning, stress response, DNA damage repair, and chromatin remodeling. Here, we show that the serine/arginine-rich protein SF2/ASF, a factor involved in splicing regulation and other RNA metabolism-related processes, is a regulator of the sumoylation pathway. The overexpression of this protein stimulates, but its knockdown inhibits SUMO conjugation. SF2/ASF interacts with Ubc9 and enhances sumoylation of specific substrates, sharing characteristics with already described SUMO E3 ligases. In addition, SF2/ASF interacts with the SUMO E3 ligase PIAS1 (protein inhibitor of activated STAT-1), regulating PIAS1-induced overall protein sumoylation. The RNA recognition motif 2 of SF2/ASF is necessary and sufficient for sumoylation enhancement. Moreover, SF2/ASF has a role in heat shock-induced sumoylation and promotes SUMO conjugation to RNA processing factors. These results add a component to the sumoylation pathway and a previously unexplored role for the multifunctional SR protein SF2/ASF. posttranslational modification | splicing factor | RNA processing | E3 ligase www.pnas.org/cgi/doi/10.1073/pnas.1004653107

RNA silencing in plants and some animals has a non-cell-autonomous effect due to an RNA signal that moves between cells or organs. To identify unique factors involved in this process, we analyzed a group of Arabidopsis mutants with defective spread of RNA silencing from a transgene expressed specifically in the phloem. These mutants accumulated reduced amounts of small interfering (si)RNA from the transgene locus and from endogenous loci TAS1, TAS2, and an inverted repeat locus IR71. The defect in TAS1 and TAS2 siRNA biogenesis is in the processing of a long siRNA precursor. We mapped the mutations to a gene encoding the Arabidopsis homolog of a protein, TEX1, which is involved in intracellular transport of RNA in animals. TEX1 is a component of the THO/TREX complex, and we show that the Arabidopsis TEX1 interacts with other predicted components of a THO/TREX complex. Correspondingly, we found at least two other components of the Arabidopsis THO core complex that are involved in RNA silencing. To reconcile the effect of these mutations on transgene and endogenous gene siRNA, we propose a mechanism in which THO/TREX processes or transports a long RNA molecule so that it can be a template for secondary siRNA production. RNA silencing | trans-acting siRNA | miRNA | inverted repeat DNA doi/ 10.1073/pnas.0911341107

Little is known about the regulation of nitrogenase genes in cyanobacteria. Transcription of the nifH1 and vnfH genes, encoding dinitrogenase reductases for the heterocyst-specific Mo-nitrogenase and the alternative V-nitrogenase, respectively, was studied by using a lacZ reporter. Despite evidence for a transcription start site just upstream of nifH1 and vnfH, promoter fragments that included these start sites did not drive the transcription of lacZ and, for nifH1, did not drive the expression of nifHDK1. Further analysis using larger regions upstream of nifH1 indicated that a promoter within nifU1 and a promoter upstream of nifB1 both contributed to expression of nifHDK1, with the nifB1 promoter contributing to most of the expression. Similarly, while the region upstream of vnfH, containing the putative transcription start site, did not drive expression of lacZ, the region that included the promoter for the upstream gene, ava4055, did. Characterization of the previously reported nifH1 and vnfH transcriptional start sites by 5'RACE (5' rapid amplification of cDNA ends) revealed that these 5' ends resulted from processing of larger transcripts rather than by de novo transcription initiation. The 5' positions of both the vnfH and nifH1 transcripts lie at the base of a stem-loop structure that may serve to stabilize the nifHDK1 and vnfH specific transcripts compared to the transcripts for other genes in the operons providing the proper stoichiometry for the Nif proteins for nitrogenase synthesis. doi: 10.1128/JB.00278-10

Human Cleavage Factor Im ([CFI.sub.m]) is an essential component of the pre-mRNA 3' processing complex that functions in the regulation of poly(A) site selection through the recognition of UGUA sequences upstream of the poly(A) site. Although the highly conserved 25 kDa subunit ([CFI.sub.m]25) of the [CFI.sub.m] complex possesses a characteristic [alpha]/[beta]/[alpha] Nudix fold, [CFI.sub.m]25 has no detectable hydrolase activity. Here we report the crystal structures of the human [CFI.sub.m]25 homodimer in complex with UGUAAA and UUGUAU RNA sequences. [CFI.sub.m]25 is the first Nudix protein to be reported to bind RNA in a sequence-specific manner. The UGUA sequence contributes to binding specificity through an intramolecular G'A Watson--Crick/sugar-edge base interaction, an unusual pairing previously found to be involved in the binding specificity of the SAM-Ill riboswitch. The structures, together with mutational data, suggest a novel mechanism for the simultaneous sequence-specific recognition of two UGUA elements within the pre-mRNA. Furthermore, the mutually exclusive binding of RNA and the signaling molecule [AP.sub.4]A (diadenosine tetraphosphate) by [CFI.sub.m]25 suggests a potential role for small molecules in the regulation of mRNA 3' processing. cleavage factor | CPSF5 | mRNA processing | Protein-RNA complex | RNA recognition doi/ 10.1073/pnas.1000848107

Constitutive expression of telomerase in human cells prevents the onset of senescence and crisis by maintaining telomere homeostasis. However, accumulating evidence suggests that the human telomerase reverse transcriptase catalytic subunit (TERT) contributes to cell physiology independently of its ability to elongate telomeres. Here we show that TERT interacts with the RNA component of mitochondrial RNA processing endoribonuclease (RMRP), a gene that is mutated in the inherited pleiotropic syndrome cartilage-hair hypoplasia. Human TERT and RMRP form a distinct ribonucleoprotein complex that has RNA-dependent RNA polymerase (RdRP) activity and produces double-stranded RNAs that can be processed into small interfering RNA in a Dicer (also known as DICER1)-dependent manner. These observations identify a mammalian RdRP composed of TERT in complex with RMRP., Telomerase is a ribonucleoprotein complex that elongates telomeres. Although several proteins interact with telomerase (1-4), the minimal components of active telomerase include the catalytic telomerase reverse transcriptase (TERT) and a [...]

Uridine insertion/deletion RNA editing is a unique form of post-transcriptional RNA processing that occurs in mitochondria of kinetoplastid protists. We have carried out 3D structural analyses of the core editing complex or 'L (ligase)-complex' from Leishmania tarentolae mitochondria isolated by the tandem affinity purification procedure (TAP). The purified material, sedimented at 20-25S, migrated in a blue native gel at 1 MDa and exhibited both precleaved and full-cycle gRNA-mediated U-insertion and U-deletion in vitro activities. The purified L-complex was analyzed by electron tomography to determine the extent of heterogeneity. Three-dimensional structural comparisons of individual particles in the tomograms revealed that a majority of the complexes have a similar shape of a slender triangle. An independent single-particle reconstruction, using a featureless Gaussian ball as the initial model, converged to a similar triangular structure. Another singleparticle reconstruction, using the averaged tomography structure as the initial model, yielded a similar structure. The REL1 ligase was localized on the model to the base of the apex by decoration with REL1-specific IgG. This structure should prove useful for a detailed analysis of the editing reaction. editing | electron microscopy | kinetoplast | Leishmania | trypanosome

The 3' untranslated regions (3' UTRs) of mRNAs contain cis-acting elements for posttranscriptional regulation of gene expression. Here, we report that mouse genes tend to express mRNAs with longer 3' UTRs as embryonic development progresses. This global regulation is controlled by alternative polyadenylation and coordinates with initiation of organogenesis and aspects of embryonic development, including morphogenesis, differentiation, and proliferation. Using myogenesis of C2C12 myoblast cells asa model, we recapitulated this process in vitro and found that 3' UTR lengthening is likely caused by weakening of mRNA polyadenylation activity. Because alternative 3' UTR sequences are typically longer and have higher AU content than constitutive ones, our results suggest that lengthening of 3' UTR can significantly augment posttranscriptional control of gene expression during embryonic development, such as microRNA-mediated regulation. mRNA processing | post-translational gene regulation

Comparative genomics has provided evidence for numerous conserved protein domains whose functions remain unknown. We identified a protein harboring 'domain of unknown function 860' (DUF860) as a component of group II intron ribonucleoprotein particles in maize chloroplasts. This protein, assigned the name WTF1 ('what's this factor?'), coimmunoprecipitates from chloroplast extract with group II intron RNAs, is required for the splicing of the introns with which it associates, and promotes splicing in the context of a heterodimer with the RNase III-domain protein RNC1. Both WTF1 and its resident DUF860 bind RNA in vitro, demonstrating that DUF860 is a previously unrecognized RNA-binding domain. DUF860 is found only in plants, where it is represented in a protein family comprising 14 orthologous groups in angiosperms. Most members of the DUF860 family are predicted to localize to chloroplasts or mitochondria, suggesting that proteins with this domain have multiple roles in RNA metabolism in both organelles. These findings add to emerging evidence that the coevolution of nuclear and organellar genomes spurred the evolution of diverse noncanonical RNA-binding motifs that perform organelle-specific functions. DUF860 | mitochondria | plastid

Activation-induced cytidine deaminase (AID) is essential for the class switch recombination (CSR) and somatic hypermutation (SHM) of Ig genes. Originally, AID was postulated to be an RNA-editing enzyme, because of its structural homology with a known RNA-editing enzyme, APOBEC1. In support of this idea, AID shares many of the properties of RNA-editing enzymes, including nucleocytoplasmic shuttling and a dependency on de novo protein synthesis. However, it has not been shown whether AID recognizes a specific mRNA and edits it to generate an enzyme involved in CSR or SHM. Here, we examined the association between AID and polyadenylated [[poly(A).sup.+]] RNA in vivo, using UV cross-linking coupled with a poly(A) capture method that relies on biotinylated oligo(dT) and streptavidin-conjugated beads. We found that both exogenous AID expressed in transfected CH12 cells and endogenous AID expressed in BL2 cells were associated with [poly(A).sup.+] RNA. Similar protein-[poly(A).sup.+] RNA complexes were formed by APOBEC1 and APOBEC3G. However, the interactions of all of these cytidine deaminase family members, including AID, with [poly(A).sup.+] RNA were indirect. This was expected for APOBEC1, which is known to act through an RNA-interacting cofactor, APOBEC1 complementation factor (ACF). In addition, the carboxy-terminal region of AID, which is essential for class switching, was also required for its interaction with [poly(A).sup.+] RNA. These results suggest that the CSR activity of AID requires an ACF-like cofactor that specifically interacts with the carboxy-terminal domain of AID. class switch recombination | RNA editing | UV cross-linking | APOBEC family | oligo dT trap

Serine/arginine-rich (SR) proteins are required for messenger RNA (mRNA) processing, export, surveillance, and translation. We show that in Chironomus tentans, nascent transcripts associate with multiple types of SR proteins in specific combinations. Alternative splicing factor (ASF)/SF2, SC35, 9G8, and hrp45/SRp55 are all present in Balbiani ring (BR) pre-messenger ribonucleoproteins (mRNPs) preferentially when introns appear in the pre-mRNA and when cotranscriptional splicing takes place. However, hrp45/SRp55 is distributed differently in the pre-mRNPs along the gene compared with ASF/SF2, SC35, and 9G8, suggesting functional differences. All four SR proteins are associated with the BR mRNPs during export to the cytoplasm. Interference with SC35 indicates that SC35 is important for the coordination of splicing, transcription, and 3' end processing and also for nucleocytoplasmic export. ASF/SF2 is associated with polyribosomes, whereas SC35, 9G8, and hrp45/SRp55 cosediment with monoribosomes. Thus, individual endogenous pre-mRNPs/ mRNPs bind multiple types of SR proteins during transcription, and these SR proteins accompany the mRNA and play different roles during the gene expression pathway in vivo.

The CDC73 tumor suppressor gene is mutationally inactivated in hereditary and sporadic parathyroid tumors. Its product, the Cdc73 protein, is a component of the RNA polymerase II and chromatin-associated human Paf1 complex (Paf1C). Here, we show that Cdc73 physically associates with the cleavage and polyadenylation specificity factor (CPSF) and cleavage stimulation factor (CstF) complexes that are required for the maturation of mRNA 3' ends in the cell nucleus. Immunodepletion experiments indicate that the Cdc73-CPSF-CstF complex is necessary for 3' mRNA processing in vitro. Microarray analysis of CDC73 siRNA-treated cells revealed INTS6, a gene encoding a subunit of the Integrator complex, as an in vivo Cdc73 target. Cdc73 depletion by siRNA resulted in decreased INTS6 mRNA abundance, and decreased association of CPSF and CstF subunits with the INTS6 locus. Our results suggest that Cdc73 facilitates association of 3' mRNA processing factors with actively-transcribed chromatin and support the importance of links between tumor suppression and mRNA maturation.

Gene expression is potently regulated through the action of RNA-binding proteins (RBPs) and microRNAs (miRNAs). Here, we present evidence of a miRNA regulating an RBP. The RBP HuR can stabilize and modulate the translation of numerous target mRNAs involved in cell proliferation, but little is known about the mechanisms that regulate HuR abundance. We identified two putative sites of miR-519 interaction on the HuR mRNA, one in its coding region (CR), one in its 3'-untranslated region (UTR). In several human carcinoma cell lines tested, HeLa (cervical), HCT116 and RKO (colon), and A2780 (ovarian), overexpression of a miR-519 precursor [(Pre)miR-519] reduced HuR abundance, while inhibiting miR-519 by using an antisense RNA [(AS)miR-519] elevated HuR levels. The influence of miR-519 was recapitulated using heterologous reporter constructs that revealed a greater repressive effect on the HuR CR than the HuR 3'-UTR target sequences, miR-519 did not alter HuR mRNA abundance, but reduced HuR biosynthesis, as determined by measuring nascent HuR translation and HuR mRNA association with polysomes. Modulation of miR-519 leading to altered HuR levels in turn affected the levels of proteins encoded by HuR target mRNAs. In keeping with HuR's proliferative influence, (AS)miR-519 significantly increased cell number and [[sup.3]H]-thymidine incorporation, while (Pre)miR-519 reduced these parameters. Importantly, the growth-promoting effects of (AS)miR-519 required the presence of HuR, because downregulation of HuR by RNAi dramatically suppressed its proliferative action. In sum, miR-519 represses HuR translation, in turn reducing HuR-regulated gene expression and cell division. elav | microRNA | post-transcriptional gene regulation | ribonucleoprotein complex | translational control

Having previously detailed a method for chip-based extraction of DNA (Anal Chem. 2003, 75, 1880-1886.), we describe here a microchip-based solid-phase extraction method for purification of RNA from biological samples is demonstrated. The method involves the use of silica beads as a solid phase, and the capacity of the device containing silica beads for RNA, RNA in the presence of protein, and DNA was determined. The capacity of the device for RNA binding in the presence of protein is 360 ng, which demonstrates sufficient capacity of the device for complete genetic analysis. An extraction of RNA can be performed on the device in as few as ~9 min (analytical time), a time comparable to that of a commercial extraction method, but with less reagent consumption. The microchip-based extraction is also performed in a closed system, unlike the commercial extraction method, which provides the advantage of decreased opportunity for the introduction of RNases and contaminants--essential for the sensitive RNA-based analyses presented in this work. RNA purified using the device was shown to be amplifiable using reverse transcription PCR (RT-PCR), allowing for translation of the method to the purification and subsequent amplification of biological samples. RNA was purified using the microchip-based method from neat semen, a mock semen stain, and cultured cells from a common pediatric cancer, alveolar rhabdomyosarcoma.

Trypanosomatid RNA editing is a unique process and essential for these organisms. It therefore represents a drug target for a group of protozoa that includes the causative agents for African sleeping sickness and other devastating tropical and subtropical diseases. Here, we present drug-like inhibitors of a key enzyme in the editing machinery, RNA-editing ligase 1 (REL1). These inhibitors were identified through a strategy employing molecular dynamics to account for protein flexibility. A virtual screen of the REL1 crystal structure against the National Cancer Institute Diversity Set was performed by using AutoDock4. The top 30 compounds, predicted to interact with REL1's ATP-binding pocket, were further refined by using the relaxed complex scheme (RCS), which redocks the compounds to receptor structures extracted from an explicitly solvated molecular dynamics trajectory. The resulting reordering of the ligands and filtering based on drug-like properties resulted in an initial recommended set of 8 ligands, 2 of which exhibited micromolar activity against REL1. A subsequent hierarchical similarity search with the most active compound over the full National Cancer Institute database and RCS rescoring resulted in an additional set of 6 ligands, 2 of which were confirmed as REL1 inhibitors with [IC.sub.50] values of [approximately equal to] 1 [micro]M. Tests of the 3 most promising compounds against the most closely related bacteriophage T4 RNA ligase 2, as well as against human DNA ligase III[beta], indicated a considerable degree of selectivity for RNA ligases. These compounds are promising scaffolds for future drug design and discovery efforts against these important pathogens. molecular dynamics | relaxed complex scheme | RNA ligase | African sleeping sickness | receptor flexibility

Group II intron ribozymes fold into their native structure by a unique stepwise process that involves an initial slow compaction followed by fast formation of the native state in a [Mg.sup.2+]-dependent manner. Single-molecule fluorescence reveals three distinct on-pathway conformations in dynamic equilibrium connected by relatively small activation barriers. From a most stable near-native state, the unobserved catalytically active conformer is reached. This most compact conformer occurs only transiently above 20 mM [Mg.sup.2+] and is stabilized by substrate binding, which together explain the slow cleavage of the ribozyme. Structural dynamics increase with increasing [Mg.sup.2+] concentrations, enabling the enzyme to reach its active state. multidomain RNA folding | single-molecule Forster resonance energy transfer | splicing | structural dynamics | metal ions

TrmA catalyzes S-adenosylmethionine (AdoMet)-dependent methylation of U54 in most tRNAs. We solved the structure of the Escherichia coli 5-methyluridine ([m.sup.5]U) 54 tRNA methyltransferase (MTase) TrmA in a covalent complex with a 19-nt T arm analog to 2.4-[Angstrom] resolution. Mutation of the TrmA catalytic base Glu-358 to Gln arrested catalysis and allowed isolation of the covalent TrmA--RNA complex for crystallization. The protein--RNA interface includes 6 nt of the T loop and two proximal base pairs of the stem. U54 is flipped out of the loop into the active site. A58 occupies the space of the everted U54 and is part of a collinear base stack G53-A58-G57-C56-U55. The RNA fold is different from T loop conformations in unbound tRNA or T arm analogs, but nearly identical to the fold of the RNA loop bound at the active site of the [m.sup.5]U MTase RumA. In both enzymes, this consensus fold presents the target U and the following two bases to a conserved binding groove on the protein. Outside of this fold, the RumA and TrmA substrates have completely different structures and protein interfaces. Loop residues other than the target U54 make more than half of their hydrogen bonds to the protein via sugar-phosphate moieties, accounting, in part, for the broad consensus sequence for TrmA substrates. RNA modification | substrate specificity | tRNA | x-ray crystallography

The evolution of RNA conformation with [Mg.sup.2+] concentration ([[Mg.sup.2+]]) is typically determined from equilibrium titration measurements or nonequilibrium single [[Mg.sup.2+]]-jump measurements. We study the folding of single RNA molecules in response to a series of periodic [[Mg.sup.2+]] jumps. The 260-residue catalytic domain of RNase P RNA from Bacillus stearothermophilus is immobilized in a microfluidic flow chamber, and the RNA conformational changes are probed by fluorescence resonance energy transfer (FRET). The kinetics of population redistribution after a [[Mg.sup.2+]] jump and the observed connectivity of FRET states reveal details of the folding pathway that complement and transcend information from equilibrium or single-jump measurements. FRET trajectories for jumps from [[Mg.sup.2+]] = 0.01 to 0.1 mM exhibit two-state behavior whereas jumps from 0.01 mM to 0.4 mM exhibit two-state unfolding but multistate folding behavior. RNA molecules in the low and high FRET states before the [[Mg.sup.2+]] increase are observed to undergo dynamics in two distinct regions of the free energy landscape separated by a high barrier. We describe the RNA structural changes involved in crossing this barrier as a 'hidden' degree of freedom because the changes do not alter the detected FRET value but do alter the observed dynamics. The associated memory prevents the populations from achieving their equilibrium values at the end of the 5- to 10-sec [[Mg.sup.2+]] interval, thereby creating a nonequilibrium steady-state condition. The capability of interrogating nonequilibrium steady-state RNA conformations and the adjustable period of [[Mg.sup.2+]]-jump cycles makes it possible to probe regions of the free energy landscape that are infrequently sampled in equilibrium or single-jump measurements. buffer jump | cooperativity | memory | FRET | electrostatic relaxation