Tetsuro Hirose

MENε/β noncoding RNAs are essential for structural integrity of nuclear paraspeckles

Recent transcriptome analyses have shown that thousands of noncoding RNAs (ncRNAs) are transcribed from mammalian genomes. Although the number of functionally annotated ncRNAs is still limited, they are known to be frequently retained in the nucleus, where they coordinate regulatory networks of gene expression. Some subnuclear organelles or nuclear bodies include RNA species whose identity and structural roles are largely unknown. We identified 2 abundant overlapping ncRNAs, MENε and MENβ (MENε/β), which are transcribed from the corresponding site in the multiple endocrine neoplasia (MEN) I locus and which localize to nuclear paraspeckles. This finding raises the intriguing possibility that MENε/β are involved in paraspeckle organization, because paraspeckles are, reportedly, RNase-sensitive structures. Successful removal of MENε/β by a refined knockdown method resulted in paraspeckle disintegration. Furthermore, the reassembly of paraspeckles disassembled by transcriptional arrest appeared to be unsuccessful in the absence of MENε/β. RNA interference and immunoprecipitation further revealed that the paraspeckle proteins p54/nrb and PSF selectively associate with and stabilize the longer MENβ, thereby contributing to the organization of the paraspeckle structure. The paraspeckle protein PSP1 is not directly involved in either MENε/β stabilization or paraspeckle organization. We postulate a model for nuclear paraspeckle body organization where specific ncRNAs and RNA-binding proteins cooperate to maintain and, presumably, establish the structure.

Involvement of a site‐specific trans‐acting factor and a common RNA‐binding protein in the editing of chloroplast mRNAs: development of a chloroplast in vitro RNA editing system

RNA editing in higher plant chloroplasts involves C→U conversion at ∼30 specific sites. An in vitro system supporting accurate editing has been developed from tobacco chloroplasts. Mutational analysis of substrate mRNAs derived from tobacco chloroplast psbL and ndhB mRNAs confirmed the participation of cis‐acting elements that had previously been identified in vivo. Competition analysis revealed the existence of site‐specific trans‐acting factors interacting with the corresponding upstream cis‐elements. A chloroplast protein of 25 kDa was found to be specifically associated with the cis‐element involved in psbL mRNA editing. Immunological analyses revealed that an additional factor, the chloroplast RNA‐binding protein cp31, is also required for RNA editing at multiple sites. This combination of site‐specific and common RNA‐binding proteins recognizes editing sites in chloroplasts.

Cis-acting elements and trans-acting factors for accurate translation of chloroplast psbA mRNAs: development of an in vitro translation system from tobacco chloroplasts.

Translational regulation is an important step of gene expression in chloroplasts. To analyze biochemical mechanisms of translational regulation unique to higher plant chloroplasts, an in vitro translation system has been developed from tobacco chloroplasts. Conditions for chloroplast extraction and the in vitro translation reaction have been optimized with a tobacco psbA‐lacZ fusion mRNA. The in vitro system supports accurate translation of a variety of chloroplasts mRNAs. Using a series of mutant psbA mRNAs, we showed that three elements within the 5′‐untranslated region of the mRNA are required for translation. Two of them are complementary to the 3′‐terminus of chloroplast 16S rRNA (termed RBS1 and RBS2) and the other is an AU‐rich sequence (UAAAUAAA) located between RBS1 and RBS2 and is termed the AU box. mRNA competition experiments using the in vitro translation reaction and gel mobility shift assays revealed the existence of a trans‐acting factor(s) for translation and its possible interaction with the AU box. We propose a model for the initiation of psbA translation whereby RBS1 and RBS2 bind cooperatively to the 3′‐end of 16S rRNA resulting in looping out of the AU box, which facilitates the interaction of a trans‐acting factor(s).

Both RNA editing and RNA cleavage are required for translation of tobacco chloroplast ndhD mRNA: a possible regulatory mechanism for the expression of a chloroplast operon consisting of functionally unrelated genes.

Tobacco chloroplast genes encoding a photosystem I component (psaC) and a NADH dehydrogenase subunit (ndhD) are transcribed as a dicistronic pre‐mRNA which is then cleaved into short mRNAs. An RNA protection assay revealed that the cleavage occurs at multiple sites in the intercistronic region. There are two possible initiation codons in the tobacco ndhD mRNA: the upstream AUG and the AUG created by RNA editing from the in‐frame ACG located 25 nt downstream. Using the chloroplast in vitro translation system, we found that translation begins only from the edited AUG. The extent of ACG to AUG editing is partial and depends on developmental and environmental conditions. In addition, the in vitro assay showed that the psaC/ndhD dicistronic mRNA is not functional and that the intercistronic cleavage is a prerequisite for both ndhD and psaC translation. Using a series of mutant mRNAs, we showed that an intramolecular interaction between an 8 nt sequence in the psaC coding region and its complementary 8 nt sequence in the 5′ ndhD UTR is the negative element for translation of the dicistronic mRNA. A possible mechanism in which the differential expression of the chloroplast operon consists of functionally unrelated genes is discussed.

RNA editing sites in tobacco chloroplast transcripts: editing as a possible regulator of chloroplast RNA polymerase activity

Abstract Genetic information in chloroplast DNA is sometimes altered at the transcript level by a process known as RNA editing. Sequence analysis of amplified cDNAs for 69 potential editing sites revealed 13 real editing sites in transcripts of 11 tobacco chloroplast genes. Together with those reported previously, these bring the total of edited sites observed in tobacco chloroplast transcripts to 31 (all involve C to U conversion). Alignment of sequences around the 31 editing sites revealed no obvious consensus, apart from an apparent bias for U or C at position −1 and A at position +2. Editing in tobacco rpoA mRNA restores the conserved leucine residue which is known to be important for transcriptional activation of the α subunit of E. coli RNA polymerase. Editing of this site is partial and the extent of editing depends on developmental conditions, suggesting that editing is, at least in part, involved in the regulation of chloroplast-encoded RNA polymerase activity.

Position within the host intron is critical for efficient processing of box C/D snoRNAs in mammalian cells

In mammalian cells, all small nucleolar RNAs (snoRNAs) that guide rRNA modification are encoded within the introns of host genes. A database analysis of human box C/D snoRNAs revealed conservation of their intronic location, with a preference for 70–80 nt upstream of the 3′ splice site. Transfection experiments showed that synthesis of gas5-encoded U75 and U76 snoRNAs dropped significantly for mutant constructs possessing longer or shorter spacers between the snoRNA and the 3′ splice site. However, the position of the snoRNA did not affect splicing of the host intron. Substitution mutations within the spacer indicated that the length, but not the specific sequence, is important. A in vitro system that couples pre-mRNA splicing and processing of U75 has been developed. U75 synthesis in vitro depends on its box C and D sequences and requires an appropriate spacer length. Further mutational analyses both in vivo and in vitro, with subsequent mapping of the branch points, revealed that the critical distance is from the snoRNA coding region to the branch point, suggesting synergy between splicing and snoRNA release.

THE ROLC PROMOTER OF AGROBACTERIUM RHIZOGENES RI PLASMID IS ACTIVATED BY SUCROSE IN TRANSGENIC TOBACCO PLANTS

The 5′-upstream region of the rolC gene of the Ri plasmid is expressed specifically in phloem cells of transgenic higher plants. In this study, we demonstrated that the rolC promoter is activated by sucrose in phloem cells of transgenic tobacco seedlings bearing rolC promoter-uidA chimeric fusion gene. Since the rolC promoter is not activated by sorbitol, sucrose metabolism rather than osmotic pressure exerted by the disaccharide may be responsible for induction. Thus, experiments using 5′-upstream deletion mutants, internal deletion mutants, and chimeric constructs with a heterologous promoter (−90 region of the cauliflower mosaic virus 35S promoter) were conducted to define the region of the rolC promoter involved in sucrose activation. The results indicated that a cis-acting sucrose responsive region of the rolC promoter is located between −135 and −94 by with respect to the transcription initiation site. In phloem cells, high concentrations of sucrose are encountered owing to ongoing translocation of photosynthates from source to sink tissues. Therefore, sucrose as a signal molecule may regulate the phloem-specific expression of the rolC promoter.

Translation of tobacco chloroplast rps14 mRNA depends on a Shine-Dalgarno-like sequence in the 5'-untranslated region but not on internal RNA editing in the coding region

The role of Shine‐Dalgarno‐like sequences in mRNAs from higher plant chloroplasts has not been analyzed experimentally so far. In vitro translation analysis has revealed that the Shine‐Dalgarno‐like sequence is essential for translation of tobacco chloroplast rps14 mRNA. Two RNA editing sites have been identified in the protein‐coding region of the rps14 mRNA. Editing of the second site was found to be partial and hence the partially edited transcripts are accumulated in tobacco green leaves. In vitro translation assays using the fully edited, partially edited and unedited rps14 mRNAs indicated that editing does not directly influence translational efficiency.

Occurrence of silent RNA editing in chloroplasts: its species specificity and the influence of environmental and developmental conditions.

We have identified three new C-to-U RNA editing sites, one in atpF and two in atpA transcripts from tobacco chloroplasts. Two of them lead to amino acid substitutions to restore the conserved amino acid found in the corresponding genes of other plants. However, one editing site in the atpA transcript was found to take place partially at the third base of a serine codon (CUC to CUU), thus not leading to an amino acid substitution. This is the first report of silent editing in chloroplasts. The extent of silent editing depends on plastid stage and light conditions, while editing at another site (found 4 nt upstream from the silent editing site) takes place constitutively even in non-photosynthetic cultured cells and bleached white seedlings grown in the presence of spectinomycin and streptomycin. In pea and spinach, despite a conservation in sequence, no editing at the site corresponding to the silent site in tobacco was found. This observation suggests that the silent editing detected in this study is species-specific.

A ribosomal protein gene (rpl32) from tobacco chloroplast DNA is transcribed from alternative promoters: similarities in promoter region organization in plastid housekeeping genes.

Multiple transcriptional start sites have been identified in the tobacco plastid ribosomal protein generpl32 by RNA mapping and in vitro capping techniques. A promoter with a canonical −10 Pribnow Box (P1) produces a major transcript in leaf chloroplasts. Transcription is also driven from additional promoters in non-photosynthetic plastids from heterotrophically cultured cells (BY2 line). Among them, a second promoter located downstream (P2) generates the most prominent transcript in this type of cell. The absence of typical plastid promoter motifs upstream of this site and the higher steady-state level of the P2-derived transcript in BY2 cells suggest a distinct modulation of transcription. Mobility shift experiments also seem to indicate the existence of differences in protein-DNA binding between both kinds of plastids with respect to a DNA fragment including the sequence upstream from the P2 starting site. The structure of therpl32 promoter region is discussed in relation to that of other plastid housekeeping genes encoding elements of the genetic machinery.