RNA biochemistry

This article is about the current status of the mfold package for RNA and DNA secondary structure prediction using nearest neighbor thermodynamic rules. The details of the free energy rules and of the latest version 3.0 software are described. Future plans are also discussed. The mfold software now runs on a variety of Unix platforms; specifically SGI Irix, Sun Solaris, Dec alpha Ultrix and on Linux. While the older interactive programs of version 2 still exist, they are now run by a variety of scripts that make for much easier handling. There is both a command line interface for mfold and an HTML interface that runs in a Unix environment but can be accessed by anyone with a web browser. The thermodynamic basis for the folding model is presented in detail, with references given for both specific free energy parameters and to overview articles that have summarized the state of these nearest neighbor parameters over the past dozen years. Both RNA and DNA rules are discussed, with some mention of parameters for RNA/DNA hybridization. Although the thermodynamic model has grown in complexity to accommodate new types of information, the folding algorithm has not yet incorporated some features, such as coaxial stacking of adjacent helices, and other features will probably remain too difficult or computationally expensive to implement. For this reason, a new energy calculation program has been introduced to recompute the free energies of predicted foldings to reflect the best of our knowledge. The most significant improvements in the mfold software are: 1. Folding times have been greatly reduced in recent years, partly because of faster computers and partly because of improvements in the algorithm. 2. Folding constraints have been expanded and are now implemented without the use of bonus energies that distort the results. 3. The output is significantly improved. Clear and enlargeable images of dot plots and of predicted foldings are now produced in PostScript and gif formats. Bases in structures may be annotated using different colors that reflect how well-determined they are in terms of their tendency to pair with other bases or to be singlestranded. Similarly, base pair probabilities from partition function calculations may be used for annotation. A detailed decomposition of each predicted folding into stacked pairs and loops with associated free energies is now provided. The mfold software has a variety of parameters that may be adjusted to improve the predictions. Several examples are presented to illustrate how to interpret folding results and how to adjust these parameters to obtain better results.

Efficient gene expression requires properly matured mRNAs for functional transcript translation. Several factors including the guard proteins monitor maturation and act as nuclear retention factors for unprocessed pre-mRNAs. Here we show that the guard protein Npl3 monitors 5'-capping. In its absence, uncapped transcripts resist degradation, because the Rat1-Rai1 5'-end degradation factors are not efficiently recruited to these faulty transcripts. Importantly, in npl3Δ, these improperly capped transcripts escape this quality control checkpoint and leak into the cytoplasm. Our data suggest a model in which Npl3 associates with the Rai1 bound pre-mRNAs. In case the transcript was properly capped and is thus CBC (cap binding complex) bound, Rai1 dissociates from Npl3 allowing the export factor Mex67 to interact with this guard protein and support nuclear export. In case Npl3 does not detect proper capping through CBC attachment, Rai1 binding persists and Rat1 can join this 5'-complex to degrade the faulty transcript.

Efficient gene expression requires properly matured mRNAs for functional transcript translation. Several factors including the guard proteins monitor maturation and act as nuclear retention factors for unprocessed pre-mRNAs. Here we show that the guard protein Npl3 monitors 5’-capping. In its absence, uncapped transcripts resist degradation, because the Rat1–Rai1 5’-end degradation factors are not efficiently recruited to these faulty transcripts. Importantly, in npl3Δ, these improperly capped transcripts escape this quality control checkpoint and leak into the cytoplasm. Our data suggest a model in which Npl3 associates with the Rai1 bound pre-mRNAs. In case the transcript was properly capped and is thus CBC (cap binding complex) bound, Rai1 dissociates from Npl3 allowing the export factor Mex67 to interact with this guard protein and support nuclear export. In case Npl3 does not detect proper capping through CBC attachment, Rai1 binding persists and Rat1 can join this 5’-complex t...

The notion that decapping leads irreversibly to messenger RNA (mRNA) decay was contradicted by the identification of capped transcripts missing portions of their 5 0 ends and a cytoplasmic complex that can restore the cap on uncapped mRNAs. In this study, we used accumulation of uncapped transcripts in cells inhibited for cytoplasmic capping to identify the targets of this pathway. Inhibition of cytoplasmic capping results in the destabilization of some transcripts and the redistribution of others from polysomes to nontranslating messenger ribonucleoproteins, where they accumulate in an uncapped state. Only a portion of the mRNA transcriptome is affected by cytoplasmic capping, and its targets encode proteins involved in nucleotide binding, RNA and protein localization, and the mitotic cell cycle. The 3 0 untranslated regions of recapping targets are enriched for AU-rich elements and micro-RNA binding sites, both of which function in capdependent mRNA silencing. These findings identify a cyclical process of decapping and recapping that we term cap homeostasis.

The PMR1 endonuclease was discovered in Xenopus liver and identified as a member of the large and diverse peroxidase gene family. The peroxidase genes arose from multiple duplication and rearrangement events, and their high degree of sequence similarity confounded attempts to identify human PMR1. The functioning of PMR1 in mRNA decay depends on the phosphorylation of a tyrosine in the C-terminal polysome targeting domain by c-Src. The sequences of regions that are required for c-Src binding and phosphorylation of Xenopus PMR1 were used to inform a bioinformatics search that identified two related genes as potential candidates for human PMR1: peroxidasin homolog (PXDN) and peroxidasin homolog-like (PXDNL) protein. Although each of these genes is predicted to encode a large, multidomain membrane-bound peroxidase, alternative splicing of PXDNL pre-mRNA yields a transcript whose predicted product is a 57-kDa protein with 42% sequence identity to Xenopus PMR1. Results presented here conf...

The N 7 methyl guanosine cap structure (where 'N' is the first transcribed nucleotide) was identified as a common feature of every RNA polymerase II transcript including mRNA or long noncoding RNA (lncRNA). This cap structure has been added to the nascent transcripts by sequential actions of mRNA Capping Enzyme (CE) and RNA methyl transferase (RNMT) in the nucleus. Every step of RNA metabolism including translation to decay is controlled by N7methyl guanosine cap and loss of this structure from the 5' end of mRNA leads to degradation. This concept of RNA decay following decapping has been changed after the identification of transcripts with stable uncapped ends through genome wide studies and discovery of cytoplasmic pool of CE (cCE) in different forms of life. cCE, in association with other proteins, can recap specific mRNA transcripts and thus cytoplasmic capping may act as post transcriptional regulator of the target mRNAs. Apart from mRNAs, many long noncoding RNAs (lncRNAs) undergo various post transcriptional processing in cytoplasm. Here, we aim to examine if lncRNAs are regulated by cCE. We have selected fifteen lncRNAs associated with sarcoma for our analysis in Osteosarcoma (U2OS) cell line that was previously used for characterizing cytoplasmic capping. These selected lncRNAs were examined for their nuclear-cytoplasmic distribution. 5'-RACE analysis was conducted to detect the uncapped forms of the lncRNAs in U2OS cells which showed several uncapped forms of these transcripts. The uncapped ends of few transcripts were determined by sequencing the cloned amplicons obtained from 5'RACE assay. To determine if these lncRNAs are targeted by cCE, transiently transfected U2OS cells were used where cytoplasmic capping is inhibited either by overexpressing dominant negative mutants of CE (K294A) or Nck1 (Nck1-M3). 5'-RACE analysis from these cells showed an accumulation of uncapped forms in cytoplasmic capping inhibited cells compared to control cells indicating regulation of these transcripts by cCE. Future CAGE and sequence analysis will identify molecular features for recapping in these transcripts.

The notion that decapping leads irreversibly to messenger RNA (mRNA) decay was contradicted by the identification of capped transcripts missing portions of their 5 0 ends and a cytoplasmic complex that can restore the cap on uncapped mRNAs. In this study, we used accumulation of uncapped transcripts in cells inhibited for cytoplasmic capping to identify the targets of this pathway. Inhibition of cytoplasmic capping results in the destabilization of some transcripts and the redistribution of others from polysomes to nontranslating messenger ribonucleoproteins, where they accumulate in an uncapped state. Only a portion of the mRNA transcriptome is affected by cytoplasmic capping, and its targets encode proteins involved in nucleotide binding, RNA and protein localization, and the mitotic cell cycle. The 3 0 untranslated regions of recapping targets are enriched for AU-rich elements and micro-RNA binding sites, both of which function in capdependent mRNA silencing. These findings identify a cyclical process of decapping and recapping that we term cap homeostasis.

Novel strategies have been developed for the identification of full-length cDNA using bioinformatics tools and multiplexed PCR methods. Usually, sequences are either incomplete or have missing UTR-sequences. Researchers still use the Rapid Amplification of cDNA Ends technique to obtain the full-length cDNA sequences. An oligo(dT) anchor-primer with a hairpin structure is designed for amplification of 3′ cDNA ends. Arbitrary degenerate and sequence-specific reverse primers were also developed for the amplification of 5′ cDNA ends, and tail-PCR is performed until the 5′ sequence of multi-assembled fragment reaches the exon-1 region which can be identified by aligning these fragments to reference genome database. Inhibitory and functional adapters are specially designed; adapter with phosphate can attach to full-length mRNAs with cap structure. In prokaryotes, a technique is reported to capture primary transcripts based on capping the 5′ triphosphorylated RNA. For the identification of...

Metazoan replication-dependent histone mRNAs are only present in S-phase, due partly to changes in their stability. These mRNAs end in a unique stem-loop (SL) that is required for both translation and cell-cycle regulation. Previous studies showed that histone mRNA degradation occurs through both 59/39 and 39/59 processes, but the relative contributions are not known. The 39 end of histone mRNA is oligouridylated during its degradation, although it is not known whether this is an essential step. We introduced firefly luciferase reporter mRNAs containing the histone 39 UTR SL (Luc-SL) and either a normal or hDcp2-resistant cap into S-phase HeLa cells. Both mRNAs were translated, and translation initially protected the mRNAs from degradation, but there was a lag of~40 min with the uncleavable cap compared to~8 min for the normal cap before rapid decay. Knockdown of hDcp2 resulted in a similar longer lag for Luc-SL containing a normal cap, indicating that 59/39 decay is important in this system. Inhibition of DNA replication with hydroxyurea accelerated the degradation of Luc-SL. Knockdown of terminal uridyltransferase (TUTase) 4 but not TUTase 3 slowed the decay process, but TUTase 4 knockdown had no effect on destabilization of the mRNA by hydroxyurea. Both Luc-SL and its 59 decay intermediates were oligouridylated. Preventing oligouridylation by 39-deoxyadenosine (cordycepin) addition to the mRNA slowed degradation, in the presence or absence of hydroxyurea, suggesting oligouridylation initiates degradation. The spectrum of oligouridylated fragments suggests the 39/59 degradation machinery stalls during initial degradation, whereupon reuridylation occurs.

Cap homeostasis is a cyclical process of decapping and recapping that impacts a portion of the mRNA transcriptome. The metastable uncapped forms of recapping targets redistribute from polysomes to non-translating mRNPs, and recapping is all that is needed for their return to the translating pool. Previous work identified a cytoplasmic capping metabolon consisting of capping enzyme (CE) and a 5-monophosphate kinase bound to adjacent domains of Nck1. The current study identifies the canonical cap methyltransferase (RNMT) as the enzyme responsible for guanine-N7 methylation of recapped mRNAs. RNMT binds directly to CE, and its presence in the cytoplasmic capping complex was demonstrated by pulldown assays, gel filtration and proximity-dependent biotinylation. The latter also identified the RNMT cofactor RAM, whose presence is required for cytoplasmic cap methyltransferase activity. These findings guided development of an inhibitor of cytoplasmic cap methylation whose action resulted in a selective decrease in levels of recapped mRNAs.

The dynamic properties of ribosomal protein L9 from Bacillus stearothermophilus were investigated in solution using an analysis of nitrogen-15 longitudinal and transverse relaxation rates and amide nitrogen-proton nuclear Overhauser effects. The relaxation rates of the amide nitrogen nuclei were found to be correlated with the angle between the amide nitrogen-proton bond vectors and the long axis of the protein. This directional dependence of the nuclear relaxation rates is consistent with the protein having a highly elongated shape in solution, consistent with that observed in previous X-ray crystallographic studies of the crystalline form. Analysis of the nuclear relaxation data shows that the solventexposed nine-turn alpha helix connecting the two domains has a relatively high degree of order, in contrast to the connecting helix in the similarly shaped, but functionally different, calmodulin protein. The rotational correlation times associated with the amide nitrogen atoms of the N-terminal domain are on average slightly shorter than those of the C-terminal domain and connecting helix, providing evidence that the Nterminal domain exhibits some degree of independence in tumbling, in addition to other fast internal motions. The putative RNA-binding surfaces in each of the protein domains are characterized by relatively low order parameters, indicating that these are the most¯exible regions of the molecule. Overall, the picture of the internal dynamics provided by nuclear relaxation measurements is similar to that obtained from a detailed study of amide proton exchange rates, but differs markedly from the picture provided by crystallographic temperature factors. The present study describes a molecule with unusual and complex dynamic properties, and supports a model where the protein functions as a``molecular strut'' within the ribosome.

Protein interactions with double-stranded RNA (dsRNA) are critical for many cell processes; however, in contrast to protein-dsDNA interactions, surprisingly little is known about the molecular basis of protein-dsRNA interactions. A large and diverse class of proteins that bind dsRNA do so by utilizing an~70 amino acid motif referred to as the dsRNA-binding domain (dsRBD). We have determined a 1.9 Å resolution crystal structure of the second dsRBD of Xenopus laevis RNA-binding protein A complexed with dsRNA. The structure shows that the protein spans 16 bp of dsRNA, interacting with two successive minor grooves and across the intervening major groove on one face of a primarily A-form RNA helix. The nature of these interactions explains dsRBD specificity for dsRNA (over ssRNA or dsDNA) and the apparent lack of sequence specificity. Interestingly, the dsRBD fold resembles a portion of the conserved core structure of a family of polynucleotidyl transferases that includes RuvC, MuA transposase, retroviral integrase and RNase H. Structural comparisons of the dsRBD-dsRNA complex and models proposed for polynucleotidyl transferase-nucleic acid complexes suggest that similarities in nucleic acid binding also exist between these families of proteins.

PED ⁄ PEA-15 (phosphoprotein enriched in diabetes ⁄ phosphoprotein enriched in astrocytes) [1-3] is a 130 residue protein that belongs to the death effector domain (DED) family, originally defined by the proapoptotic Fas-associated death domain protein (FADD) . The DEDs, together with the structurally related death domain (DD), caspase recruitment domain (CARD) and pyrin domain (PYD), are members of the death motif superfamily characterized by a conserved six a-helix bundle structure . Death motif proteins mediate homotypic interactions whereby CARD-CARD, DD-DD, PYD-PYD and DED-DED contacts are established and play a critical role in the formation of oligomeric signaling complexes, such as the death-inducing signaling complex, the apoptosome and the PIDDosome [9].

PED ⁄ PEA-15 (phosphoprotein enriched in diabetes ⁄ phosphoprotein enriched in astrocytes) [1-3] is a 130 residue protein that belongs to the death effector domain (DED) family, originally defined by the proapoptotic Fas-associated death domain protein (FADD) . The DEDs, together with the structurally related death domain (DD), caspase recruitment domain (CARD) and pyrin domain (PYD), are members of the death motif superfamily characterized by a conserved six a-helix bundle structure . Death motif proteins mediate homotypic interactions whereby CARD-CARD, DD-DD, PYD-PYD and DED-DED contacts are established and play a critical role in the formation of oligomeric signaling complexes, such as the death-inducing signaling complex, the apoptosome and the PIDDosome [9].

The notion that decapping leads irreversibly to messenger RNA (mRNA) decay was contradicted by the identification of capped transcripts missing portions of their 5 0 ends and a cytoplasmic complex that can restore the cap on uncapped mRNAs. In this study, we used accumulation of uncapped transcripts in cells inhibited for cytoplasmic capping to identify the targets of this pathway. Inhibition of cytoplasmic capping results in the destabilization of some transcripts and the redistribution of others from polysomes to nontranslating messenger ribonucleoproteins, where they accumulate in an uncapped state. Only a portion of the mRNA transcriptome is affected by cytoplasmic capping, and its targets encode proteins involved in nucleotide binding, RNA and protein localization, and the mitotic cell cycle. The 3 0 untranslated regions of recapping targets are enriched for AU-rich elements and micro-RNA binding sites, both of which function in capdependent mRNA silencing. These findings identify a cyclical process of decapping and recapping that we term cap homeostasis.

Specific recognition of double-stranded RNA (dsRNA) by dsRNAbinding domains (dsRBDs) is involved in a large number of biological and regulatory processes. Although structures of dsRBDs in complex with dsRNA have revealed how they can bind to dsRNA in general, these do not explain how a dsRBD can recognize specific RNAs. Rnt1p, a member of the RNase III family of dsRNA endonucleases, is a key component of the Saccharomyces cerevisiae RNA-processing machinery. The Rnt1p dsRBD has been implicated in targeting this endonuclease to its RNA substrates, by recognizing hairpins closed by AGNN tetraloops. We report the solution structure of Rnt1p dsRBD complexed to the 5 terminal hairpin of one of its small nucleolar RNA substrates, the snR47 precursor. The conserved AGNN tetraloop fold is retained in the protein-RNA complex. The dsRBD contacts the RNA at successive minor, major, and tetraloop minor grooves on one face of the helix. Surprisingly, neither the universally conserved G nor the highly conserved A are recognized by specific hydrogen bonds to the bases. Rather, the N-terminal helix fits snugly into the minor groove of the RNA tetraloop and top of the stem, interacting in a non-sequencespecific manner with the sugar-phosphate backbone and the two nonconserved tetraloop bases. Mutational analysis of residues that contact the tetraloop region show that they are functionally important for RNA processing in the context of the entire protein in vivo. These results show how a single dsRBD can convey specificity for particular RNA targets, by structure specific recognition of a conserved tetraloop fold.

Rnt1 endoribonuclease, the yeast homolog of RNAse III, plays an important role in the maturation of a diverse set of RNAs. The enzymatic activity requires a conserved catalytic domain, while RNA binding requires the doublestranded RNA-binding domain (dsRBD) at the C-terminus of the protein. While bacterial RNAse III enzymes cleave double-stranded RNA, Rnt1p specifically cleaves RNAs that possess short irregular stem-loops containing 12-14 base pairs interrupted by internal loops and bulges and capped by conserved AGNN tetraloops. Consistent with this substrate specificity, the isolated Rnt1p dsRBD and the 30-40 amino acids that follow bind to AGNN-containing stemloops preferentially in vitro. In order to understand how Rnt1p recognizes its cognate processing sites, we have defined its minimal RNA-binding domain and determined its structure by solution NMR spectroscopy and X-ray crystallography. We observe a new carboxy-terminal helix following a canonical dsRBD structure. Removal of this helix reduces binding to Rnt1p substrates. The results suggest that this helix allows the Rnt1p dsRBD to bind to short RNA stem-loops by modulating the conformation of helix a1, a key RNA-recognition element of the dsRBD.

One of the most common structural motifs in RNA-binding proteins is the RNA-binding domain (RBD). These domains share a common a/b sandwich tertiary fold, and are highly conserved, though they bind diverse RNA targets with a wide range of binding af®nities. The N-terminal RNA-binding domain (RBD1) of the human U1A protein binds speci®cally to stem/loop II of the U1 snRNA with sub-nanomolar af®nity. Solvent-exposed aromatic residues on the b-sheet surface are highly conserved among RBD domains; in RBD1, these are Tyr13 and Phe56, with a unique Gln at position 54. Effects of substitutions at these positions were examined using energetic pairwise coupling to describe the communication between these residues in both the free and RNA-bound states of the protein. 15 N NMR experiments were used to determine effects of the b-sheet substitutions on the structural and dynamic properties of this domain. The combination of thermodynamic pairwise coupling and 15 N-backbone dynamics provides direct evidence for local cooperative interactions among Y13, Q54, and F56, and a non-conserved loop that directly affect RNA-binding. The results describe how conserved and non-conserved regions of an RBD can communicate with each other to mediate recognition of the RNA.

We propose a (3, 2)D CT-HCCH-COSY experiment to rapidly collect the data and provide significant dispersion in the spectral region containing 13 C-1 H cross peaks of CH 3 groups belonging to Ala, Ile, Leu, Met, Thr and Val residues. This enables one to carry out chemical shift based editing and grouping of all the 13 C-1 H cross peaks of CH 3 groups belonging to Ala, Ile, Leu, Met, Thr and Val residues in fractionally (10%) 13 C-labelled proteins, which in turn aids in the sequence-specific resonance assignments in general and side-chain resonance assignments in particular, in any given protein. Further, we demonstrate the utility of this experiment for stereospecific assignments of the pro-R and pro-S methyl groups belonging to the Leu and Val residues in fractionally (10%) 13 C-labelled proteins. The proposed experiment opens up a wide range of applications in resonance assignment strategies and structure determination of proteins.

The human Protein Kinase R (PKR) is one of the important and critical components of the innate immune response against viral infection. It regulates distinct cellular functions and controls the fate of an RNA molecule in the cell. PKR dephosphorylation is characterised by inhibitory interactions between the kinase domain and the RNA binding domains (RBDs), but the complete structural details and dynamics of the latent state and its activation are not yet clear. Several studies on the activation of double stranded RNA binding domain (dsRBD) of Protein Kinase R (dsRBD-PKR) have been attempted during the last few decades. In order to further investigate on its activation mechanism, we have analysed the backbone [ 1 H-15 N]-dynamics and chemical shift perturbation studies of dsRBD-PKR in the presence of bacteriophage Pf1. Pf1 is known to orient the protein in the presence of external magnetic field and, can be further used to get useful information about the protein properties such as the different dynamic behaviour in multidomain proteins like dsRBD-PKR. These NMR based results improve our basic understanding of the domain flexibilities of dsRBD-PKR where some secondary structural regions of dsRBM1 (dsRNA binding motif 1) show a different behaviour than the one from dsRBM2 (dsRNA binding motif 2) these observations further lead to a better understanding of PKR mechanism where dsRBM1 has more affinity towards RNA as compared to dsRBM2.

Double-stranded RNA binding domains of human protein kinase R (dsRBD-PKR) regulate distinct cellular functions and the fate of an RNA molecule in the cell. This highly homologous domains present in multiple copies in a number of species, exhibit individual and specific functional specificity. Number of NMR and X-ray crystallographic structural studies reveals that such domains take a common ␣-␤-␤-␤-␣ tertiary fold. However, the functional specificities of these domains could be due to the dynamics of the individual amino acid residues, as has been shown earlier in the case of backbone dynamics of 15 N-1 H of dsRNA binding motifs (dsRBMs) of human protein kinase R (PKR) (Nanduri S, Rahman F, Williams BRG, Qin J. EMBO J 2000;19:5567-5574). To further investigate if the differences in dynamics of the two dsRBMs are restricted to only backbone, or if the side-chain motions are also different to the extent of influencing their packing of the two hydrophobic cores, we have investigated the methyl group dynamics using 13 C-methyl relaxation measurements. The results show that the hydrophobic core of dsRBM1 is more tightly packed than dsRBM2, and it seems to undergo less fast scale motions in the subnanosecond regime. Proteins 2006;62:501-508.