Stable Stem-loop Structure Research Articles

Tumor Suppressor Protein Pdcd4 Inhibits Translation of p53 mRNA

The tumor suppressor protein Pdcd4 is thought to suppress translation of mRNAs containing structured 5'-UTRs by interacting with translation initiation factor eIF4A and inhibiting its helicase activity. However, natural target mRNAs regulated by Pdcd4 so far are mostly unknown. Here, we identified p53 mRNA as a translational target of Pdcd4. We found that Pdcd4 is associated with p53 mRNA and suppresses its translation. The inhibitory effect of Pdcd4 on the translation of p53 mRNA depends on the ability of Pdcd4 to interact with eIF4A and is mediated by the 5'-UTR of p53 mRNA, which is able to form a stable stem-loop structure. We show that treatment of cells with DNA-damaging agents decreases the expression of Pdcd4. This suggests that translational suppression by Pdcd4 plays a role in maintaining a low level of p53 in unstressed cells and that this suppression is abrogated due to low levels of Pdcd4 after DNA damage. Overall, our work demonstrates for the first time that Pdcd4 is directly involved in translational suppression of a natural mRNA with a 5'-structured UTR and provides novel insight into the translational control of p53 expression.

Regulation of zinc-responsive Slc39a5 (Zip5) translation is mediated by conserved elements in the 3′-untranslated region

Translation of the basolateral zinc transporter ZIP5 is repressed during zinc deficiency but Zip5 mRNA remains associated with polysomes and can be rapidly translated when zinc is repleted. Herein, we examined the mechanisms regulating translation of Zip5. The 3′-untranslated region (UTR) of Zip5 mRNA is well conserved among mammals and is predicted by mFOLD to form a very stable stem-loop structure. Three algorithms predict this structure to be flanked by repeated seed sites for miR-328 and miR-193a. RNAse footprinting supports the notion that a stable stem-loop structure exists in this 3′-UTR and electrophoretic mobility shift assays detect polysomal protein(s) binding specifically to the stem-loop structure in the Zip5 3′-UTR. miR-328 and miR-193a are expressed in tissues known to regulate Zip5 mRNA translation in response to zinc availability and both are polysome-associated consistent with Zip5 mRNA localization. Transient transfection assays using native and mutant Zip5 3′-UTRs cloned 3′ to luciferase cDNA revealed that the miRNA seed sites and the stem-loop function together to augment translation of Zip5 mRNA when zinc is replete.Electronic supplementary materialThe online version of this article (doi:10.1007/s10534-011-9508-4) contains supplementary material, which is available to authorized users.

The large intergenic region of Rice tungro bacilliform virus evolved differentially among geographically distinguished isolates

Rice tungro bacilliform virus (RTBV) is a plant pararetrovirus. The large intergenic region (LIGR) of RTBV having a single transcriptional promoter produces more than genome length pregenomic RNA (pgRNA) which directs synthesis of circular double-stranded viral DNA and serves as a polycistronic mRNA. By computer-aided analysis of LIGR, the 11 RTBV isolates sequenced so far were compared with respect to structural organization of promoter and pgRNA 5'-leader. The results revealed only 74.90% identity at LIGR between 'Southeast Asian' (SEA) and 'South Asian' (SA) isolates of RTBV indicating considerable variation between two groups which was also reflected during analysis of promoter and leader sequence. The predicted promoter region of SA isolates exhibited major variations in terms of transcription start site and consensus sequences of cis motifs expecting further exploitation of promoter region of SA isolates. The reduced length of leader sequence along with less numbers and different arrangements of small open reading frames (sORFs) in case of SA isolates might have some alterations in the control of expression of ORF II and III between the two groups. In spite of these variations, the leader sequence of both SEA and SA type isolates showed formation of stable secondary or stem-loop structure having identical features for efficient translation. The conservation of sORF1 at seven nucleotides upstream of stable stem-loop, CU-rich sequence following the sORF1 stop codon and AU-rich shunt landing sequence immediately downstream of the secondary structure suggested conservation of ribosomal shunt mechanism in all RTBV isolates irrespective of their geographical distribution.

Mitochondrial genome sequences of the striped field mice Apodemus agrarius coreae and Apodemus agrarius chejuensis

We determined the complete mitochondrial (mt) genome sequences of the striped field mice Apodemus agrarius coreae and Apodemus agrarius chejuensis. The mt genomes of A. a. coreae and A. a. chejuensis are 16,260 and 16,261 base pairs in length, respectively. The general features of the 13 protein-coding genes of the two species are similar to those of other rodents. The TAG termination codon for NADH dehydrogenase subunit (ND) 3 is unique to Apodemus in the Muroidea. The L-strand replication origin has the potential to form a stable stem–loop structure. Within the control region, a termination-associated sequence and several conserved sequence blocks were observed. The diversity of the 13 protein-coding genes, 2 rRNAs, and 1 control region between the two species ranged between 0.005 (ATP8) and 0.027 (ND4L).

Organization and variation of mitochondrial DNA control region in pleurodiran turtles

Three complete mitochondrial DNA (mtDNA) control regions (CRs) of Chelodina rugosa (Ogilby, 1890), Chelus fimbriata (Schneider, 1783), and Podocnemis unifilis (Troschel, 1848) were firstly determined using Long-PCR method and the length were 1,016 bp, 1,149 bp, and 985bp, respectively. Together with CRs of Pelomedusa subrufa (Bonnaterre, 1789) and nearly complete CR of Podocnemis expansa (Schweigger, 1812) obtained from GenBank, the structural and evolutionary characteristics of mtDNA CRs in pleurodiran turtle were analyzed in this study. We identified three functional domains (TAS, CD, and CSB domains) as well as their conservation sequences (TAS, CSB-F, and CSB-1) according to their homology to those of other turtles. Within the TAS domain, an interrupted poly-C stretch was found in C. rugosa, C. fimbriata, and P. subrufa, which also exists in the published mt DNA CRs of Chrysemys picta (Schneider, 1783), Trachemys scripta (Thunberg in Schoepff, 1792), and Trionyx triunguis (Forskål, 1775). The analysis of the origin for the poly-C sequences in TAS domain from six turtles suggested that the poly-C sequences are more related to "goose hairpin" in birds rather than CSB2 in CSB domain. In the CSB domain, CSB2 and CSB3, which were determined in CRs of Cryptodira, were absent in Pleurodira CRs, indicating the regulative mechanisms of transcription may be varied in both two suborders and the lack of CSB2 and CSB3 could be proposed as one of diagnostic characters between Pleurodira and Cryptodira at molecular level. As for CR of other cryptodiran turtles, variable number of tandem repeats (VNTRs) in the 3' end of the CRs was found in the five pleurodiran turtles. Interestingly, the long repeated motifs from each species could form stable stem-loop secondary structures, suggesting that the repeated sequences may play an important role in regulating replication of the mitochondrial genome in Pleurodiran, and the secondary structures of VNTRs may provide some potential information in phylogenetic inference.

The mitochondrial genome of Apodemus peninsulae (Rodentia, Muridae)

The complete mitochondrial (mt) genome of the Korean field mouse Apodemus peninsulae was sequenced and found to be 16,266 bp in length. The mt protein-coding genes of A. peninsulae had ATG, GTG, ATC, and ATA as initiation codons and TAA, TAG, TA, and T as termination codons. Two forms each of trnL and trnS and the three tRNA clusters, IQM, WANCY, and HSL were identified, as in the typical Rodentia mt genome. Among tRNAs, abnormal cloverleaf structure of trnS(AGY) was identified in DHU arm. The l-strand replication origin has the potential to form a stable stem–loop structure and control region has several conserved sequence elements.

Stem‐Loop RT‐qPCR for miRNAs

This unit presents a specific and sensitive quantitative reverse-transcription PCR (RT-qPCR) method for measuring individual microRNAs (miRNAs) in tissue or cultured cells. miRNAs are 17 to 24 nucleotides (nt) in length. Standard and quantitative PCR methods require a template that is at least two times the length of either of the specific forward or reverse primers, each typically ∼ 20 nt in length. Thus, the target minimum length is ≥ 40 nt, making miRNAs too short for standard RT-qPCR methods. In this assay, each of the RT-qPCR nucleic acid reagents, including the RT-primer, the forward and reverse PCR primers, and the hydrolysis probe, contain design features that, together, optimize miRNA specificity and assay sensitivity. The RT-primer contains a highly stable stem-loop structure that lengthens the target cDNA. The forward PCR primer adds additional length with nucleotides that optimize its melting temperature (T(m)) and enhance assay specificity. The reverse primer disrupts the stem loop. Assay specificity is further optimized by placement of the probe over much of the original miRNA sequence, and the probe T(m) is optimized by addition of a minor groove binding (MGB) moiety.

Identification of microRNAs in ecological model plant <i>Mimulus</i>

MicroRNAs (miRNAs) are small, non-coding and regulatory RNAs about 20 to 24 nucleotides long. Their conserved nature among the various organisms makes them a good source of new miRNAs discovery by comparative genomics approach. This study resulted in 34 miRNAs belonging to 21 families in ecological model plant Mimulus. All the miRNA families (mir 156, 157, 159, 160, 164, 172, 319, 393, 395, 397, 399, 400, 403, 417, 419, 472, 782, 854, 858, 867 and 2112) are found for the first time in Mimulus. The MIR-399 is found as precursor miRNA cluster with 4 mature sequences. All 34 miRNA precursors form stable minimum free energy stem loop structure as their orthologues form and the mature miRNAs reside in the stem portion of the stem loop structure. Twenty eight are from Mimulus guttatus and six miRNAs belong to Mimulus lewisii. Their targets consist of dihy-droflavonol-4-reductase, cycloidea-like protein, DNA-directed RNA polymerase II, maturase (matR) and transcription factors like; squamosa-promoter binding, MYB, palmate-like pentafoliata 1.

Tethering of Poly(A)-binding Protein Interferes with Non-translated mRNA Decay from the 5′ End in Yeast*

The decay of eukaryotic mRNA is triggered mainly by deadenylation, which leads to decapping and degradation from the 5' end of an mRNA. Poly(A)-binding protein has been proposed to inhibit the decapping process and to stabilize mRNA by blocking the recruitment of mRNA to the P-bodies where mRNA degradation takes place after stimulation of translation initiation. In contrast, several lines of evidence show that poly(A)-binding protein (Pab1p) has distinct functions in mRNA decay and translation in yeast. To address the translation-independent function of Pab1p in inhibition of decapping, we examined the contribution of Pab1p to the stability of non-translated mRNAs, an AUG codon-less mRNA or an mRNA containing a stable stem-loop structure at the 5'-UTR. Tethering of Pab1p stabilized non-translated mRNAs, and this stabilization did not require either the eIF4G-interacting domain of Pab1p or the Pab1p-interacting domain of eIF4G. In a ski2Δ mutant in which 3' to 5' mRNA degradation activity is defective, stabilization of non-translated mRNAs by the tethering of Pab1p lacking an eIF4G-interacting domain (Pab1-34Cp) requires a cap structure but not a poly(A) tail. In wild type cells, stabilization of non-translated mRNA by tethered Pab1-34Cp results in the accumulation of deadenylated mRNA. These results strongly suggest that tethering of Pab1p may inhibit the decapping reaction after deadenylation, independent of translation. We propose that Pab1p inhibits the decapping reaction in a translation-independent manner in vivo.

Mitochondrial genome of the cotton bollworm Helicoverpa armigera (Lepidoptera: Noctuidae) and comparison with other Lepidopterans

We present the complete sequence of the mitochondrial genome (mitogenome) of the cotton bollworm Helicoverpa armigera. The 15,347-bp mitogenome of H. armigera was arranged in the same order described for all other sequenced lepidopterans, which differs from the most common type found in insects, due to the movement of trnM to a position 5′-upstream of trnI. The gene overlap in the H. armigera mitogenome is totally 23 bp in six locations. The H. armigera mitogenome has a total of 175 bp of intergenic spacer sequences spread over 14 regions ranging in size from 1 to 45 bp. The nucleotide composition of the whole mitogenome of H. armigera is highly A+T biased, accounting for 80.97%, with a slightly positive AT skewness and negative GC skewness, indicating the occurrence of more A than T, C more than G. The protein-encoding genes have typical mitochondrial start codons, except for cox1, which contains the unusual CGA. The cox1, cox2, and nad4 genes have incomplete stop codons (T). The lrRNA and srRNA genes are 1395 and 794-bp long, respectively. All tRNAs have a typical cloverleaf structure of mitochondrial tRNAs, except for trnS1(AGN), the dihydrouridine arm of which could not form a stable stem-loop structure. The H. armigera A+T-rich region contains a conserved structure combining the motif ATAGA and a 19-bp poly-T stretch, but absence of the 9-bp poly-A element upstream of trnM.

Novel cis-active structures in the coding region mediate CRM1-dependent nuclear export of IFN-α 1 mRNA

We recently reported the chromosome region maintenance 1 (CRM1)-dependent nuclear export of intron-less human interferon-α1 (IFN-α1) mRNA, which encodes a main effecter of host innate immunity. We show that the coding region of IFN-α1 mRNA forms novel secondary structures that are responsible for the CRM1-dependent export of the transcript. Deletion-mutagenesis, in vivo export assays, and computer analyses of the folding potentials of export-competent fragments revealed the presence of a domain, termed the conserved secondary structure (CSS), comprising two adjacent putative stable stem-loop structures (nt 208-452). Internal deletion-mutagenesis and constitutive export assays of each stem-loop structure demonstrated that subregions 308-322 and 352-434 act as a core element by conferring the export function on the CSS. Leptomycin B (LMB) inhibition of the CRM1 pathway decreased the export of core element RNA, implying that the principal site of CRM1 action for exporting IFN-α1 mRNA resides within the core element. An RNPS1 (RNA-binding protein S1, serine-rich domain) cDNA was isolated by yeast three-hybrid screening, using bait containing two CSS regions. We showed that RNPS1 might recognize IFN-α1 mRNP that includes CRM1. The data demonstrate that interaction between RNA structures in the coding region and CRM1 affects the nucleocytoplasmic translocation of IFN-α1 mRNA.

Interaction between Thymidylate Synthase and Its Cognate mRNA in Zebrafish Embryos

Thymidylate synthase (TS), which catalyzes the de novo synthesis of dUMP, is an important target for cancer therapy. In this report, the effects of 5-fluorouracil (5-FU) and ZD1694 on the regulation of TS gene expression were evaluated in zebrafish embryos. Our results revealed that the expression of TS was increased by about six-fold when embryos were treated with 1.0 µM 5-FU and there was a greater than 10-fold increase in the TS protein level after treatment with 0.4 µM ZD1694. Northern blot analysis confirmed that expression of TS mRNA was identical in treated or untreated embryos. Gel shift and immunoprecipitation assays revealed that zebrafish TS was specifically bound with its cognate mRNA in vitro and in vivo. We identified a 20 nt RNA sequence, TS:N20, localized to the 5′-UTR of TS mRNA, which corresponded to nt 13–32; TS:N20 bound to the TS protein with an affinity similar to that of the full-length TS mRNA. The MFold program predicted that TS:N20 formed a stable stem-loop structure similar to that of the cis-acting element found in human TS mRNA. Variant RNAs with either a deletion or mutation in the core motif of TS:N20 were unable to bind to the TS protein. In vitro translation experiments, using the rabbit lysate system, confirmed that zebrafish TS mRNA translation was significantly repressed when an excess amount of TS protein was included in the system. Additionally, a TS stability experiment confirmed that treatment of zebrafish embryos with 5-FU could increase the TS stability significantly, and the half life of TS protein was about 2.7 times longer than in untreated embryos. Our study revealed a structural requirement for the interaction of TS RNA with TS protein. These findings also demonstrated that the increase in TS protein induced by 5-FU occurs at the post-transcriptional level and that increased stability and translation efficiency both contributed to the increase in TS protein levels induced by TS inhibitors.

Abstract 3163: Posttranscriptional regulation of androgen receptor mRNA by an ErbB3 binding protein 1 (EBP1) in prostate cancer

Abstract Introduction: Regulation of AR mRNA stability and translation, two central processes that control AR expression, is little known. ErbB3 binding protein 1 (EBP1) overexpression decreases AR-target genes including AR itself at the mRNA and protein levels. Interestingly, AR protein levels were down regulated to a greater extent than mRNA levels. EBP1 has recently been demonstrated to be an RNA binding protein. We therefore examined the ability of EBP1 to regulate AR post-transcriptionally. Experimental procedure: The stability of AR mRNA was analyzed in LNCaP cells engineered with EBP1 cDNA or shRNA at different times after treatment with actinomycin D by measuring the remaining levels of AR and GAPDH mRNAs by RT-qPCR. The steady-state levels of AR mRNA and protein were also detected by quantitative RT-PCR and immunoblotting analysis. To assess the association of endogenous EBP1 and AR mRNA, LNCaP cell lysates were immunoprecipitated with antibodies recognizing EBP1 or with control IgG, followed by PCR amplification using primers specific for AR. The interaction between the biotinylated transcripts spanning a 397-nt region of the 3’UTR containing the AR UC-rich element, or CAG polyglutamine repeat, which is predicted to form a stable stem-loop structure, at the 5’-end of the coding sequence in the AR mRNA, and recombinant His-EBP1 fusion proteins was assessed by biotin pull-down assays followed by Western blot analysis. In similar way, the domain of EBP1 responsible for the binding to CAG and UC-rich sequences was mapped. To determine if EBP1 affects the ability of the UC-rich region to accelerate AR mRNA decay, a pGL3-Luc-UC plasmid was constructed by fusing the UC-rich region in the 3’UTR of AR mRNA to the 3’ end of the Firefly-luciferase coding sequence and luciferase reporter assays performed in LNCaP cells engineered with EBP1 cDNA or shRNA. To test if EBP1 may modulate not only AR stability, but also its translation, linear sucrose gradient fractionation was performed to investigate the distribution of AR mRNA associated with polysomes in LNCaP cells engineered with EBP1 cDNA or shRNA. A summary of the data: EBP1 promoted AR mRNA decay through physical interaction with a conserved UC-rich motif within the 3’-UTR of AR. The ability of EBP1 to accelerate AR mRNA decay was further enhanced by HRG treatment. EBP1 also bound to a CAG-formed stem-loop in the 5’ coding region of AR mRNA and reduced percentage of AR mRNA in a translationally active polysome pool. Conclusion: Our current work indentified EBP1 as a novel AR mRNA-binding protein regulating AR mRNA stability and translation, and suggests that targeting EBP1 has potential mechanistic and functional significance in the therapeutic management of prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3163.

The mitochondrial genome of the butterfly Papilio xuthus (Lepidoptera: Papilionidae) and related phylogenetic analyses

The nearly complete mitochondrial genome of the butterfly Papilio xuthus (Lepidoptera: Papilionidae) was sequenced for its nucleotide sequence of 13,964 bp. The genome has a typical gene order identical to other lepidopteran species. All tRNAs showed same stable canonical clover-leaf structure as those of other insects, except for tRNA(Ser) (AGN), in which the dihydrouracil arm (DHU arm) could not form stable stem-loop structure. Anomalous initiation codons have been observed for the cox1 gene, where the ATTACG hexa-nucleotide was believed to be involved in the initiation signaling. Twelve mitochondrial protein-coding gene sequence data were used to infer the phylogenetic relationships among the insect orders. Even though the number of insect orders represented by complete mitochondrial genomes is still limited, several well-established relationships are evident in the phylogenetic analysis of the complete sequences. Monophyly of the Homometabola was not supported in this paper. Phylogenetic analyses of the available species of Bombycoidea, Pyraloidea, Papilionoidea and Tortricidea bolstered the current morphology-based hypothesis that Bombycoidea, Pyraloidea and Papilionoidea are monophyletic (Obtectomera). Bombycoidea (Bombyx mandarina and Antheraea pernyi) and Papilionoidea (P. xuthus and Coreana raphaelis) formed a sister group.

Computational Design of an RNA Nanoparticle Consisting of a Three-Way Junction and pre-miRNAs

Recent research on RNA-based regulation, RNAi, and development of human diseases has shed light on the huge potential of miRNAs as novel therapeutic agents for medicine. It has been suggested that a successful tissue-targetable nucleic acid delivery system has to overcome the problem of poor stability of nucleic acids in biological media. To solve this problem we rationally designed a pre-miRNA-nanoparticle-mediated RNA delivery system by integrating computer modeling, miRNA regulatory function and RNA structure versatility. It has been well documented that the initial product of a miRNA, the pri-miRNA, is transcribed in the nucleus. It forms a highly stable stem-loop structure that is processed to form the pre-miRNA by the RNase III enzyme, Drosha. The stable pre-miRNA stem-loop structure of 60-70 nt is transported to the cytoplasm and is then processed into a short double stranded fragment by dicer. Finally the miRNA duplex is unwound to form a ∼22-nt single stranded mature miRNA which is associated with the RISC complex. In this study, we present a computational design of a synthetic, highly stable superstructure made from an RNA junction that can accommodate multiple pre-miRNAs. A three-way junction building block, which was obtained from the RNAjunction database, was attached to three human let-7 pre-miRNAs. This highly stable RNA nanoparticle is expected to enable the binding of the dicer protein for the efficient cleavage of the pre-miRNA in the cytoplasm. As a result, the mature let-7 miRNAs can enter into the targeted cell cytosol, and be protected from degrading interactions while performing its specific regulatory functions.

The complete mitochondrial genome of the fall webworm, Hyphantria cunea (Lepidoptera: Arctiidae)

The complete mitochondrial genome (mitogenome) of the fall webworm, Hyphantria cunea (Lepidoptera: Arctiidae) was determined. The genome is a circular molecule 15 481 bp long. It presents a typical gene organization and order for completely sequenced lepidopteran mitogenomes, but differs from the insect ancestral type for the placement of tRNA(Met). The nucleotide composition of the genome is also highly A + T biased, accounting for 80.38%, with a slightly positive AT skewness (0.010), indicating the occurrence of more As than Ts, as found in the Noctuoidea species. All protein-coding genes (PCGs) are initiated by ATN codons, except for COI, which is tentatively designated by the CGA codon as observed in other lepidopterans. Four of 13 PCGs harbor the incomplete termination codon, T or TA. All tRNAs have a typical clover-leaf structure of mitochondrial tRNAs, except for tRNA(Ser)(AGN), the DHU arm of which could not form a stable stem-loop structure. The intergenic spacer sequence between tRNA(Ser)(AGN) and ND1 also contains the ATACTAA motif, which is conserved across the Lepidoptera order. The H. cunea A+T-rich region of 357 bp is comprised of non-repetitive sequences, but harbors several features common to the Lepidoptera insects, including the motif ATAGA followed by an 18 bp poly-T stretch, a microsatellite-like (AT)(8) element preceded by the ATTTA motif, an 11 bp poly-A present immediately upstream tRNA(Met). The phylogenetic analyses support the view that the H. cunea is closerly related to the Lymantria dispar than Ochrogaster lunifer, and support the hypothesis that Noctuoidea (H. cunea, L. dispar, and O. lunifer) and Geometroidea (Phthonandria atrilineata) are monophyletic. However, in the phylogenetic trees based on mitogenome sequences among the lepidopteran superfamilies, Papillonoidea (Artogeia melete, Acraea issoria, and Coreana raphaelis) joined basally within the monophyly of Lepidoptera, which is different to the traditional classification.

Rationally designed aptamer-based fluorescence polarization sensor dedicated to the small target analysis

A direct fluorescence polarization (FP) assay strategy, dedicated to the small molecule sensing and based on the unique induced-fit binding mechanism of end-labelled nucleic acid aptamers, has been recently developed by our group. Small target binding has been successfully converted into a significant increase of the fluorescence anisotropy signal presumably produced by the reduction of the local motional freedom of the dye. In order to generalize the approach, a rational FP sensor methodology was established herein, by engineering instability in the secondary structure of an aptameric recognition element. The anti-adenosine DNA aptamer, labelled by a single fluorescein dye at its 3′ extremity, was employed as a model functional nucleic acid probe. The terminal stem of the stem-loop structure was shortened to induce a destabilized/denatured conformation which promoted the local segmental mobility of the dye and then a significant depolarization process. Upon target binding, the structural change of the aptamer induced the formation of a stable stem-loop structure, leading to the reduction of the dye mobility and the increase in the fluorescence anisotropy signal. This reasoned approach was applied to the sensing of adenosine and adenosine monophosphate and their chiral analysis.

A moss pentatricopeptide repeat protein binds to the 3′ end of plastid clpP pre‐mRNA and assists with mRNA maturation

Pentatricopeptide repeat (PPR) proteins constitute a large family in land plants and are required for various post-transcriptional steps associated with RNA in plant organelles. The moss Physcomitrella patens PPR protein, PpPPR_38, is a nuclear-encoded chloroplast protein and was previously shown to be involved in the maturation step of chloroplast clpP pre-mRNA. To understand precisely the molecular function of PpPPR_38, we prepared recombinant PpPPR_38 protein and characterized it in maturation steps of clpP pre-mRNA. In vitro RNA-binding assays showed that the recombinant protein strongly bound to the clpP-5'-rps12 intergenic region, which is highly AU-rich and includes an inverted repeat sequence potentially forming a stem-loop structure. Digestion of the bound RNA region by RNase V1 was significantly accelerated by the addition of the recombinant protein. This strongly suggests that the binding of PpPPR_38 facilitates the formation of a stable stem-loop structure. An in vitro degradation assay using chloroplast lysates gave rise to the possibility that the stable stem-loop structure formed by PpPPR_38 contributes the correct intergenic RNA cleavage and protection of mature clpP mRNA against 3' to 5' exoribonuclease. Because an RNA-binding assay also showed weak binding to the clpP first exon-intron region, PpPPR_38 is likely to be related to the splicing of clpP pre-mRNA. Taking together all of the above findings, we conclude that PpPPR_38 is necessary for several steps in the clpP mRNA maturation process.

Three discontinuous loop nucleotides in the 3′ terminal stem-loop are required for Red clover necrotic mosaic virus RNA-2 replication

The genome of Red clover necrotic mosaic virus (RCNMV) consists of positive-sense, single-stranded RNA-1 and RNA-2. The 29 nucleotides at the 3' termini of both RNAs are nearly identical and are predicted to form a stable stem-loop (SL) structure, which is required for RCNMV RNA replication. Here we performed a systematic mutagenesis of the RNA-2 3' SL to identify the nucleotides critical for replication. Infectivity and RNA replication assays indicated that the secondary structure of the 3' SL and its loop sequence UAUAA were required for RNA replication. Single-nucleotide substitution analyses of the loop further pinpointed three discontinuous nucleotides (L1U, L2A, and L4A) that were vital for RNA replication. A 3-D model of the 3' SL predicted the existence of a pocket formed by these three nucleotides that could be involved in RNA-protein interaction. The functional groups of the bases participating in this interaction at these positions are discussed.

5′ and 3′ ends of chloroplast transcripts can both be stabilised by protein ‘caps’: a new model for polycistronic RNA maturation

5′ and 3′ ends of chloroplast transcripts can both be stabilised by protein ‘caps’: a new model for polycistronic RNA maturation