RNA current issue

[REVIEW] Modulating the activity of the peptidyl transferase center of the ribosome

Beringer, M. — 2008-04-21

The peptidyl transferase (PT) center of the ribosome catalyzes two nucleophilic reactions, peptide bond formation between aminoacylated tRNA substrates and, together with release factor, peptide release. Structure and function of the PT center are modulated by binding of aminoacyl-tRNA or release factor, thus providing the basis for the specificity of catalysis. Another way by which the function of the PT center is controlled is signaling from the peptide exit tunnel. The SecM nascent peptide induces ribosome stalling, presumably by inhibition of peptide bond formation. Similarly, the release factor-induced hydrolytic activity of the PT center can be suppressed by the TnaC nascent peptide contained in the exit tunnel. Thus, local and long-range conformational rearrangements can lead to changes in the reaction specificity and catalytic activity of the PT center.

[REVIEW] Splicing regulation: From a parts list of regulatory elements to an integrated splicing code

Wang, Z., Burge, C. B. — 2008-04-21

Alternative splicing of pre-mRNAs is a major contributor to both proteomic diversity and control of gene expression levels. Splicing is tightly regulated in different tissues and developmental stages, and its disruption can lead to a wide range of human diseases. An important long-term goal in the splicing field is to determine a set of rules or "code" for splicing that will enable prediction of the splicing pattern of any primary transcript from its sequence. Outside of the core splice site motifs, the bulk of the information required for splicing is thought to be contained in exonic and intronic cis-regulatory elements that function by recruitment of sequence-specific RNA-binding protein factors that either activate or repress the use of adjacent splice sites. Here, we summarize the current state of knowledge of splicing cis-regulatory elements and their context-dependent effects on splicing, emphasizing recent global/genome-wide studies and open questions.

[BIOINFORMATICS] Dual coding of siRNAs and miRNAs by plant transposable elements

Piriyapongsa, J., Jordan, I. K. — 2008-04-21

We recently proposed a specific model whereby miRNAs encoded from short nonautonomous DNA-type TEs known as MITEs evolved from corresponding ancestral full-length (autonomous) elements that originally encoded short interfering (siRNAs). Our miRNA-origins model predicts that evolutionary intermediates may exist as TEs that encode both siRNAs and miRNAs, and we analyzed Arabidopsis thaliana and Oryza sativa (rice) genomic sequence and expression data to test this prediction. We found a number of examples of individual plant TE insertions that encode both siRNAs and miRNAs. We show evidence that these dual coding TEs can be expressed as readthrough transcripts from the intronic regions of spliced RNA messages. These TE transcripts can fold to form the hairpin (stem–loop) structures characteristic of miRNA genes along with longer double-stranded RNA regions that typically are processed as siRNAs. Taken together with a recent study showing Drosha independent processing of miRNAs from Drosophila introns, our results indicate that ancestral miRNAs could have evolved from TEs prior to the full elaboration of the miRNA biogenesis pathway. Later, as the specific miRNA biogenesis pathway evolved, and numerous other expressed inverted repeat regions came to be recognized by the miRNA processing endonucleases, the host gene-related regulatory functions of miRNAs emerged. In this way, host genomes were afforded an additional level of regulatory complexity as a by-product of TE defense mechanisms. The siRNA-to-miRNA evolutionary transition is representative of a number of other regulatory mechanisms that evolved to silence TEs and were later co-opted to serve as regulators of host gene expression.

[REPORT] The aptamer core of SAM-IV riboswitches mimics the ligand-binding site of SAM-I riboswitches

2008-04-21

A novel family of riboswitches, called SAM-IV, is the fourth distinct set of mRNA elements to be reported that regulate gene expression via direct sensing of S-adenosylmethionine (SAM or AdoMet). SAM-IV riboswitches share conserved nucleotide positions with the previously described SAM-I riboswitches, despite rearranged structures and nucleotide positions with family-specific nucleotide identities. Sequence analysis and molecular recognition experiments suggest that SAM-I and SAM-IV riboswitches share similar ligand binding sites, but have different scaffolds. Our findings support the view that RNA has considerable structural versatility and reveal that riboswitches exploit this potential to expand the scope of RNA in genetic regulation.

[REPORT] Two molecular features contribute to the Argonaute specificity for the microRNA and RNAi pathways in C. elegans

Jannot, G., Boisvert, M.-E. L., Banville, I. H., Simard, M. J. — 2008-04-21

In Caenorhabditis elegans, specific Argonaute proteins are dedicated to the RNAi and microRNA pathways. To uncover how the precise Argonaute selection occurs, we designed dsRNA triggers containing both miRNA and siRNA sequences. While dsRNA carrying nucleotides mismatches can only enter the miRNA pathway, a fully complementary dsRNA successfully rescues let-7 miRNA function and initiates silencing by RNAi. We demonstrated that RDE-1 is essential for RNAi induced by the perfectly paired trigger, yet is not required for silencing by the let-7 miRNA. In contrast, ALG-1/ALG-2 are required for the miRNA function, but not for the siRNA-directed gene silencing. Finally, a dsRNA containing a bulged miRNA and a perfectly paired siRNA can enter both pathways suggesting that the sorting of small RNAs occurs after that the dsRNA trigger has been processed by Dicer. Thus, our data suggest that the selection of Argonaute proteins is affected by two molecular features: (1) the structure of the small RNA duplex; and (2) the Argonautes specific characteristics.

[REPORT] Microarray-based analysis of stress-regulated microRNAs in Arabidopsis thaliana

Liu, H.-H., Tian, X., Li, Y.-J., Wu, C.-A., Zheng, C.-C. — 2008-04-21

High-salinity, drought, and low temperature are three common environmental stress factors that seriously influence plant growth and development worldwide. Recently, microRNAs (miRNAs) have emerged as a class of gene expression regulators that have also been linked to stress responses. However, the relationship between miRNA expression and stress responses is just beginning to be explored. Here, we identified 14 stress-inducible miRNAs using microarray data in which the effects of three abiotic stresses were surveyed in Arabidopsis thaliana. Among them, 10 high-salinity-, four drought-, and 10 cold-regulated miRNAs were detected, respectively. miR168, miR171, and miR396 responded to all of the stresses. Expression profiling by RT-PCR analysis showed great cross-talk among the high-salinity, drought, and cold stress signaling pathways. The existence of stress-related elements in miRNA promoter regions provided further evidence supporting our results. These findings extend the current view about miRNA as ubiquitous regulators under stress conditions.

[ARTICLE] Normalization of microRNA expression levels in quantitative RT-PCR assays: Identification of suitable reference RNA targets in normal and cancerous human solid tissues

Peltier, H. J., Latham, G. J. — 2008-04-21

Proper normalization is a critical but often an underappreciated aspect of quantitative gene expression analysis. This study describes the identification and characterization of appropriate reference RNA targets for the normalization of microRNA (miRNA) quantitative RT-PCR data. miRNA microarray data from dozens of normal and disease human tissues revealed ubiquitous and stably expressed normalization candidates for evaluation by qRT-PCR. miR-191 and miR-103, among others, were found to be highly consistent in their expression across 13 normal tissues and five pair of distinct tumor/normal adjacent tissues. These miRNAs were statistically superior to the most commonly used reference RNAs used in miRNA qRT-PCR experiments, such as 5S rRNA, U6 snRNA, or total RNA. The most stable normalizers were also highly conserved across flash-frozen and formalin-fixed paraffin-embedded lung cancer tumor/NAT sample sets, resulting in the confirmation of one well-documented oncomir (let-7a), as well as the identification of novel oncomirs. These findings constitute the first report describing the rigorous normalization of miRNA qRT-PCR data and have important implications for proper experimental design and accurate data interpretation.

[ARTICLE] Experimental validation of the importance of seed complement frequency to siRNA specificity

2008-04-21

Pairing between the hexamer seed region of a small interfering RNA (siRNA) guide strand (nucleotides 2–7) and complementary sequences in the 3' UTR of mature transcripts has been implicated as an important element in off-target gene regulation and false positive phenotypes. To better understand the association between seed sequences and off-target profiles we performed an analysis of all possible (4096) hexamers and identified a nonuniform distribution of hexamer frequencies across the 3' UTR transcriptome. Subsequent microarray analysis of cells transfected with siRNAs having seeds with low, medium, or high seed complement frequencies (SCFs) revealed that duplexes with low SCFs generally induced fewer off-targets and off-target phenotypes than molecules with more abundant 3' UTR complements. These findings provide the first experimentally validated strategy for designing siRNAs with enhanced specificity and allow for more accurate interpretation of high throughput screening data generated with existing siRNA/shRNA collections.

[ARTICLE] Lack of secondary structure characterizes the 5' ends of mammalian mitochondrial mRNAs

Jones, C. N., Wilkinson, K. A., Hung, K. T., Weeks, K. M., Spremulli, L. L. — 2008-04-21

The mammalian mitochondrial genome encodes 13 proteins, which are synthesized at the direction of nine monocistronic and two dicistronic mRNAs. These mRNAs lack both 5' and 3' untranslated regions. The mechanism by which the specialized mitochondrial translational apparatus locates start codons and initiates translation of these leaderless mRNAs is currently unknown. To better understand this mechanism, the secondary structures near the start codons of all 13 open reading frames have been analyzed using RNA SHAPE chemistry. The extent of structure in these mRNAs as assessed experimentally is distinctly lower than would be predicted by current algorithms based on free energy minimization alone. We find that the 5' ends of all mitochondrial mRNAs are highly unstructured. The first 35 nucleotides for all mitochondrial mRNAs form structures with free energies less favorable than –3 kcal/mol, equal to or less than a single typical base pair. The start codons, which lie at the very 5' ends of these mRNAs, are accessible within single stranded motifs in all cases, making them potentially poised for ribosome binding. These data are consistent with a model in which the specialized mitochondrial ribosome preferentially allows passage of unstructured 5' sequences into the mRNA entrance site to participate in translation initiation.

[ARTICLE] miR-148 targets human DNMT3b protein coding region

Duursma, A. M., Kedde, M., Schrier, M., le Sage, C., Agami, R. — 2008-04-21

MicroRNAs (miRNAs) are small noncoding RNA molecules of 20–24 nucleotides that regulate gene expression. In animals, miRNAs form imperfect interactions with sequences in the 3' Untranslated region (3'UTR) of mRNAs, causing translational inhibition and mRNA decay. In contrast, plant miRNAs mostly associate with protein coding regions. Here we show that human miR-148 represses DNA methyltransferase 3b (Dnmt3b) gene expression through a region in its coding sequence. This region is evolutionary conserved and present in the Dnmt3b splice variants Dnmt3b1, Dnmt3b2, and Dnmt3b4, but not in the abundantly expressed Dnmt3b3. Whereas overexpression of miR-148 results in decreased DNMT3b1 expression, short-hairpin RNA-mediated miR-148 repression leads to an increase in DNMT3b1 expression. Interestingly, mutating the putative miR-148 target site in Dnmt3b1 abolishes regulation by miR-148. Moreover, endogenous Dnmt3b3 mRNA, which lacks the putative miR-148 target site, is resistant to miR-148-mediated regulation. Thus, our results demonstrate that the coding sequence of Dnmt3b mediates regulation by the miR-148 family. More generally, we provide evidence that coding regions of human genes can be targeted by miRNAs, and that such a mechanism might play a role in determining the relative abundance of different splice variants.

[ARTICLE] Sm protein methylation is dispensable for snRNP assembly in Drosophila melanogaster

Gonsalvez, G. B., Praveen, K., Hicks, A. J., Tian, L., Matera, A. G. — 2008-04-21

Sm proteins form stable ribonucleoprotein (RNP) complexes with small nuclear (sn)RNAs and are core components of the eukaryotic spliceosome. In vivo, the assembly of Sm proteins onto snRNAs requires the survival motor neurons (SMN) complex. Several reports have shown that SMN protein binds with high affinity to symmetric dimethylarginine (sDMA) residues present on the C-terminal tails of SmB, SmD1, and SmD3. This post-translational modification is thought to play a crucial role in snRNP assembly. In human cells, two distinct protein arginine methyltransferases (PRMT5 and PRMT7) are required for snRNP biogenesis. However, in Drosophila, loss of Dart5 (the fruit fly PRMT5 ortholog) has little effect on snRNP assembly, and homozygous mutants are completely viable. To resolve these apparent differences, we examined this topic in detail and found that Drosophila Sm proteins are also methylated by two methyltransferases, Dart5/PRMT5 and Dart7/PRMT7. Unlike dart5, we found that dart7 is an essential gene. However, the lethality associated with loss of Dart7 protein is apparently unrelated to defects in snRNP assembly. To conclusively test the requirement for sDMA modification of Sm proteins in Drosophila snRNP assembly, we constructed a fly strain that exclusively expresses an isoform of SmD1 that cannot be sDMA modified. Interestingly, these flies were viable, and snRNP assays revealed no defects in comparison to wild type. In contrast, dart5 mutants displayed a strong synthetic lethal phenotype in the presence of a hypomorphic Smn mutation. We therefore conclude that dart5 is required for viability when SMN is limiting.

[ARTICLE] Identification of TTP mRNA targets in human dendritic cells reveals TTP as a critical regulator of dendritic cell maturation

2008-04-21

Dendritic cells provide a critical link between innate and adaptive immunity and are essential to prime a naive T-cell response. The transition from immature dendritic cells to mature dendritic cells involves numerous changes in gene expression; however, the role of post-transcriptional changes in this process has been largely ignored. Tristetraprolin is an AU-rich element mRNA-binding protein that has been shown to regulate the stability of a number of cytokines and chemokines of mRNAs. Using TTP immunoprecipitations and Affymetrix GeneChips, we identified 393 messages as putative TTP mRNA targets in human dendritic cells. Gene ontology analysis revealed that ~25% of the identified mRNAs are associated with protein synthesis. We also identified six MHC Class I alleles, five MHC Class II alleles, seven chemokine and chemokine receptor genes, indoleamine 2,3 dioxygenase, and CD86 as putative TTP ligands. Real-time PCR was used to validate the GeneChip data for 15 putative target genes and functional studies performed for six target genes. These data establish that TTP regulates the expression of DUSP1, IDO, SOD2, CD86, and MHC Class I-B and F via the 3'-untranslated region of each gene. A novel finding is the demonstration that TTP can interact with and regulate the expression of non-AU-rich element-containing messages. The data implicate TTP as having a broader role in regulating and limiting the immune response than previously suspected.

[ARTICLE] Hairpin RNAs derived from RNA polymerase II and polymerase III promoter-directed transgenes are processed differently in plants

2008-04-21

RNA polymerase III (Pol III) as well as Pol II (35S) promoters are able to drive hairpin RNA (hpRNA) expression and induce target gene silencing in plants. siRNAs of 21 nt are the predominant species in a 35S Pol II line, whereas 24- and/or 22-nucleotide (nt) siRNAs are produced by a Pol III line. The 35S line accumulated the loop of the hpRNA, in contrast to full-length hpRNA in the Pol III line. These suggest that Pol II and Pol III-transcribed hpRNAs are processed by different pathways. One Pol III transgene produced only 24-nt siRNAs but silenced the target gene efficiently, indicating that the 24-nt siRNAs can direct mRNA degradation; specific cleavage was confirmed by 5' rapid amplification of cDNA ends (RACE). Both Pol II- and Pol III-directed hpRNA transgenes induced cytosine methylation in the target DNA. The extent of methylation is not correlated with the level of 21-nt siRNAs, suggesting that they are not effective inducers of DNA methylation. The promoter of a U6 transgene was significantly methylated, whereas the promoter of the endogenous U6 gene was almost free of cytosine methylation, suggesting that endogenous sequences are more resistant to de novo DNA methylation than are transgene constructs.

[ARTICLE] Unexpected roles for UPF1 in HIV-1 RNA metabolism and translation

2008-04-21

The HIV-1 ribonucleoprotein (RNP) contains the major structural protein, pr55^Gag, viral genomic RNA, as well as the host protein, Staufen1. In this report, we show that the nonsense-mediated decay (NMD) factor UPF1 is also a component of the HIV-1 RNP. We investigated the role of UPF1 in HIV-1-expressing cells. Depletion of UPF1 by siRNA resulted in a dramatic reduction in steady-state HIV-1 RNA and pr55^Gag. Pr55^Gag synthesis, but not the cognate genomic RNA, was efficiently rescued by expression of an siRNA-insensitive UPF1, demonstrating that UPF1 positively influences HIV-1 RNA translatability. Conversely, overexpression of UPF1 led to a dramatic up-regulation of HIV-1 expression at the RNA and protein synthesis levels. The effects of UPF1 on HIV-1 RNA stability were observed in the nucleus and cytoplasm and required ongoing translation. We also demonstrate that the effects exerted by UPF1 on HIV-1 expression were dependent on its ATPase activity, but were separable from its role in NMD and did not require interaction with UPF2.

[ARTICLE] Maturation of the 5S rRNA 5' end is catalyzed in vitro by the endonuclease tRNase Z in the archaeon H. volcanii

2008-04-21

Ribosomal RNA molecules are synthesized as precursors that have to undergo several processing steps to generate the functional rRNA. The 5S rRNA in the archaeon Haloferax volcanii is transcribed as part of a multicistronic transcript containing both large rRNAs and one or two tRNAs. Release of the 5S rRNA from the precursor requires two endonucleolytic cleavages by enzymes as yet not identified. Here we report the first identification of an archaeal 5S rRNA processing endonuclease. The enzyme tRNase Z, which was initially identified as tRNA processing enzyme, generates not only tRNA 3' ends but also mature 5S rRNA 5' ends in vitro. Interestingly, the sequence upstream of the 5S rRNA can be folded into a mini-tRNA, which might explain the processing of this RNA by tRNase Z. The endonuclease is active only at low salt concentrations in vitro, which is in contrast to the 2–4 M KCl concentration present inside the cell in vivo. Electron microscopy studies show that there are no compartments inside the Haloferax cell that could provide lower salt environments. Processing of the 5S rRNA 5' end is not restricted to the haloarchaeal tRNase Z since tRNase Z enzymes from a thermophilic archaeon, a lower and a higher eukaryote, are as well able to cleave the tRNA-like structure 5' of the 5S rRNA. Knock out of the tRNase Z gene in Haloferax volcanii is lethal, showing that the protein is essential for the cell.

[ARTICLE] An important role of G638 in the cis-cleavage reaction of the Neurospora VS ribozyme revealed by a novel nucleotide analog incorporation method

Jaikaran, D., Smith, M. D., Mehdizadeh, R., Olive, J., Collins, R. A. — 2008-04-21

We describe a chemical coupling procedure that allows joining of two RNAs, one of which contains a site-specific base analog substitution, in the absence of divalent ions. This method allows incorporation of nucleotide analogs at specific positions even into large, cis-cleaving ribozymes. Using this method we have studied the effects of substitution of G638 in the cleavage site loop of the VS ribozyme with a variety of purine analogs having different functional groups and pK_a values. Cleavage rate versus pH profiles combined with kinetic solvent isotope experiments indicate an important role for G638 in proton transfer during the rate-limiting step of the cis-cleavage reaction.

[ARTICLE] The AU-rich element mRNA decay-promoting activity of BRF1 is regulated by mitogen-activated protein kinase-activated protein kinase 2

Maitra, S., Chou, C.-F., Luber, C. A., Lee, K.-Y., Mann, M., Chen, C.-Y. — 2008-04-21

Regulated mRNA decay is a highly important process for the tight control of gene expression. Inherently unstable mRNAs contain AU-rich elements (AREs) in the 3' untranslated regions that direct rapid mRNA decay by interaction with decay-promoting ARE-binding proteins (ARE-BPs). The decay of ARE-containing mRNAs is regulated by signaling pathways that are believed to directly target ARE-BPs. Here, we show that BRF1 involved in ARE-mediated mRNA decay (AMD) is phosphorylated by MAPK-activated protein kinase 2 (MK2). In vitro kinase assays using different BRF1 fragments suggest that MK2 phosphorylates BRF1 at four distinct sites, S54, S92, S203, and an unidentified site at the C terminus. Coexpression of an active form of MK2 inhibits ARE mRNA decay activity of BRF1. MK2-mediated inhibition of BRF1 requires phosphorylation at S54, S92, and S203. Phosphorylation of BRF1 by MK2 does not appear to alter its ability to interact with AREs or to associate with mRNA decay enzymes. Thus, MK2 inhibits BRF1-dependent AMD through direct phosphorylation. Although the mechanism underlying this inhibition is still unclear, it appears to target BRF1-dependent AMD at a level downstream from RNA binding and the recruitment of mRNA decay enzymes.

[ARTICLE] Cap-dependent eukaryotic initiation factor-mRNA interactions probed by cross-linking

Lindqvist, L., Imataka, H., Pelletier, J. — 2008-04-21

Cap-dependent ribosome recruitment to eukaryotic mRNAs during translation initiation is stimulated by the eukaryotic initiation factor (eIF) 4F complex and eIF4B. eIF4F is a heterotrimeric complex composed of three subunits: eIF4E, a 7-methyl guanosine cap binding protein; eIF4A, a DEAD-box RNA helicase; and eIF4G. The interactions of eIF4E, eIF4A, and eIF4B with mRNA have previously been monitored by chemical- and UV-based cross-linking approaches aimed at characterizing the initial protein/mRNA interactions that lead to ribosome recruitment. These studies have led to a model whereby eIF4E interacts with the 7-methyl guanosine cap structure in an ATP-independent manner, followed by an ATP-dependent interaction of eIF4A and eIF4B. Herein, we apply a splint-ligation-mediated approach to generate 4-thiouridine-containing mRNA adjacent to a radiolabel group that we utilize to monitor cap-dependent cross-linking of proteins adjacent to, and downstream from, the cap structure. Using this approach, we demonstrate interactions between eIF4G, eIF4H, and eIF3 subunits with the mRNA during the cap recognition process.

[ARTICLE] TbRGG1, an essential protein involved in kinetoplastid RNA metabolism that is associated with a novel multiprotein complex

Hashimi, H., Zikova, A., Panigrahi, A. K., Stuart, K. D., Lukes, J. — 2008-04-21

The uridine insertion/deletion RNA editing of kinetoplastid mitochondrial transcripts is performed by complex machinery involving a number of proteins and multiple protein complexes. Here we describe the effect of silencing of TbRGG1 gene by RNA interference on RNA editing in procyclic stage of Trypanosoma brucei. TbRGG1 is an essential protein for cell growth, the absence of which results in an overall decline of edited mRNAs, while the levels of never-edited RNAs remain unaltered. Repression of TbRGG1 expression has no effect on the 20S editosome and MRP1/2 complex. TAP-tag purification of TbRGG1 coisolated a novel multiprotein complex, and its association was further verified by TAP-tag analyses of two other components of the complex. TbRGG1 interaction with this complex appears to be mediated by RNA. Our results suggest that the TbRGG1 protein functions in stabilizing edited RNAs or editing efficiency and that the associated novel complex may have a role in mitochondrial RNA metabolism. We provisionally name it putative mitochondrial RNA-binding complex 1 (put-MRB complex 1).

[METHOD] Selection of peptides interfering with a ribosomal frameshift in the human immunodeficiency virus type 1

Dulude, D., Theberge-Julien, G., Brakier-Gingras, L., Heveker, N. — 2008-04-21

The human immunodeficiency virus of type 1 (HIV-1) uses a programmed -1 ribosomal frameshift to produce the precursor of its enzymes, and changes in frameshift efficiency reduce replicative fitness of the virus. We used a fluorescent two-reporter system to screen for peptides that reduce HIV-1 frameshift in bacteria, knowing that the frameshift can be reproduced in Escherichia coli. Expression of one reporter, the green fluorescent protein (GFP), requires the HIV-1 frameshift, whereas the second reporter, the red fluorescent protein (RFP), is used to assess normal translation. A peptide library biased for RNA binding was inserted into the sequence of the protein thioredoxin and expressed in reporter-containing bacteria, which were then screened by fluorescence-activated cell sorting (FACS). We identified peptide sequences that reduce frameshift efficiency by over 50% without altering normal translation. The identified sequences are also active against different frameshift stimulatory signals, suggesting that they bind a target important for frameshifting in general, probably the ribosome. Successful transfer of active sequences to a different scaffold in a eukaryotic test system demonstrates that the anti-frameshift activity of the peptides is neither due to scaffold-dependent conformation nor effects of the scaffold protein itself on frameshifting. The method we describe identifies peptides that will provide useful tools to further study the mechanism of frameshift and may permit the development of lead compounds of therapeutic interest.