Varda Liveanu

The sequence and structure of the 3′-untranslated regions of chloroplast transcripts are important determinants of mRNA accumulation and stability

A general characteristic of the 3′-untranslated regions (3′ UTRs) of plastid mRNAs is an inverted repeat (IR) sequence that can fold into a stem-loop structure. These stem-loops are RNA 3′-end processing signals and determinants of mRNA stability, not transcription terminators. Incubation of synthetic RNAs corresponding to the 3′ UTRs of Chlamydomonas chloroplast genes atpB and petD with a chloroplast protein extract resulted in the accumulation of stable processing products. Synthetic RNAs of the petA 3′ UTR and the antisense strand of atpB 3′ UTR were degraded in the extract. To examine 3′ UTR function in vivo, the atpB 3′ UTR was replaced with the 3′ UTR sequences of the Chlamydomonas chloroplast genes petD, petD plus trnR, rbcL, petA and E. coli thrA by biolistic transformation of Chlamydomonas chloroplasts. Each 3′ UTR was inserted in both the sense and antisense orientations. The accumulation of both total atpB mRNA and ATPase β-subunit protein in all transformants was increased compared to a strain in which the atpB 3′ UTR had been deleted. However, the level of discrete atpB transcripts in transformants containing the antisense 3′ UTR sequences was reduced to approximately one-half that of transformants containing the 3′ UTRs in the sense orientation. These results imply that both the nucleotide sequences and the stem-loop structures of the 3′ UTRs are important for transcript 3′-end processing, and for accumulation of the mature mRNAs.

RNA Polyadenylation in Prokaryotes and Organelles; Different Tails Tell Different Tales

The addition of poly(A) tails to RNA is a phenomenon common to almost all organisms examined as of today. In eukaryotes, a stable poly(A) tail is added to the 3′ end of most nuclear-encoded mRNAs. This process is important for mRNA stability and translation initiation. In addition, polyadenylation of nuclear-encoded transcripts in yeast was recently reported to promote RNA degradation. In prokaryotes and organelles, RNA molecules are polyadenylated as part of a polyadenylation-dependent RNA degradation mechanism. This process consists sequentially of endonucleolytic cleavage, addition of degradation-inducing poly(A)-rich sequences to these cleavage products, and exonucleolytic degradation. In spinach chloroplasts the latter two steps, polyadenylation and exonucleolytic degradation, are performed by a single phosphorolytic and processive enzyme, polynucleotide phosphorylase (PNPase), while there is no equivalent to the E. coli poly(A)-polymerase enzyme. This was also found to be the case in cyanobacteria, a prokaryote believed to be related to the evolutionary ancestor of the chloroplast, and also in several other bacteria. No RNA polyadenylation was detected in the halophilic archaea Haloferax volcanii, which lacks the exosome complex, or in yeast mitochondria, which lack PNPase. Unlike other organelles, mammalian mitochondrial transcripts are known to include stable poly(A) tails at their 3′ ends, much like the case of nuclear-encoded mRNA. However, recent data have revealed that in addition to full-length, stably polyadenylated transcripts, nonabundant, truncated, polyadenylated RNA fragments are present in human mitochondria. These results suggest that the polyadenylation-dependent RNA degradation pathway is present in human mitochondria together with the addition of stable poly(A) tails at the mature 3′ end. We describe a possible scenario illustrating the evolution of RNA polyadenylation and its related functions found in bacteria, archaea, organelles, and eukaryotes. Referee: Dr. David Stern, Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, NY 14853.

Growth at acidic pH induces an amastigote stage-specific protein in Leishmania promastigotes

This paper constitutes the first investigation into the effect of pH on phenotypic expression in Leishmania. The advantage of our experimental system is that it makes use of the extracellular (promastigote) form, which can be readily grown in culture medium, as a model to study parasite development in vivo

RNA-Binding Characteristics of a Ribonucleoprotein from Spinach Chloroplast

A chloroplast (nuclear-encoded) RNA-binding protein (28RNP) was previously purified from spinach (Spinacia oleracea). This 28RNP was found to be the major RNA-binding protein co-purified during the isolation scheme of 3[prime] end RNA-processing activity of several chloroplastic genes. To learn more about the possible involvement of 28RNP in the 3[prime] end RNA-processing event, we investigated the RNA-binding properties and the location of the protein in the chloroplast. We found that recombinant Escherichia coliexpressed 28RNP binds with apparently the same affinity to every chloroplastic 3[prime] end RNA that was analyzed, as well as to RNAs derived from the 5[prime] end or the coding region of some chloroplastic genes. Differences in the RNA-binding affinities for some chloroplastic 3[prime] end RNAs were observed when the recombinant 28RNP was compared with the “native” 28RNP in the chloroplast-soluble protein extract. In addition, we found that the 28RNP is not associated with either thylakoid-bound or soluble polysomes in which a great portion of the chloroplast rRNA and mRNA are localized. These results suggest that the native 28RNP binds specifically to certain RNA molecules in the chloroplast in which other components (possibly proteins) and/or posttranslational modifications are involved in determining RNA-binding specificity of the 28RNP.

Tricyclic drugs reduce proton motive force in Leishmania donovani promastigotes.

Tricyclic compounds have been suggested as potential anti-leishmanial drugs. We have studied the effect of tricyclic drugs on several cellular functions in L. donovani promastigotes. Imipramine inhibits proline transport and reduces delta pH and cellular ATP at relatively high concentrations (IC50 = 50-80 microM). High concentrations of imipramine are also required to kill L. donovani promastigotes (LD50 greater than 50 microM). The presence of a chlorine atom in the side ring of either imipramine or promazine results in a three-fold increase in both IC50 and LD50 values. Tricyclic compounds in which the nitrogen in the middle ring was substituted with a carbon atom (amitryptyline and chlorprothixene) are most effective in causing cell death and in decreasing proline transport and delta pH (IC50 congruent to 5 microM), whereas depletion of cellular ATP requires a higher drug concentration (IC50 = 12 microM). Transchlorprothixene has IC50 values for proline transport, delta pH and cellular ATP that are similar to those of amitriptyline, whereas the cis isomer is less active. Imipramine, chlomipramine and chlorpromazine decrease the membrane potential in promastigotes. There is a direct correlation between inhibition of membrane transport of proline and the size of the membrane potential at various concentrations of the drugs. Taken together, the multiple effects of the tricyclic drugs on cellular functions in Leishmania suggest that the drugs cause cellular death by non-specific mechanisms, probably involving a general increase in membrane permeability.

Hybrid bio-photo-electro-chemical cells for solar water splitting

Photoelectrochemical water splitting uses solar power to decompose water to hydrogen and oxygen. Here we show how the photocatalytic activity of thylakoid membranes leads to overall water splitting in a bio-photo-electro-chemical (BPEC) cell via a simple process. Thylakoids extracted from spinach are introduced into a BPEC cell containing buffer solution with ferricyanide. Upon solar-simulated illumination, water oxidation takes place and electrons are shuttled by the ferri/ferrocyanide redox couple from the thylakoids to a transparent electrode serving as the anode, yielding a photocurrent density of 0.5 mA cm−2. Hydrogen evolution occurs at the cathode at a bias as low as 0.8 V. A tandem cell comprising the BPEC cell and a Si photovoltaic module achieves overall water splitting with solar to hydrogen efficiency of 0.3%. These results demonstrate the promise of combining natural photosynthetic membranes and man-made photovoltaic cells in order to convert solar power into hydrogen fuel.

Identification of LACTB2, a metallo-β-lactamase protein, as a human mitochondrial endoribonuclease

Abstract Post-transcriptional control of mitochondrial gene expression, including the processing and generation of mature transcripts as well as their degradation, is a key regulatory step in gene expression in human mitochondria. Consequently, identification of the proteins responsible for RNA processing and degradation in this organelle is of great importance. The metallo-β-lactamase (MBL) is a candidate protein family that includes ribo- and deoxyribonucleases. In this study, we discovered a function for LACTB2, an orphan MBL protein found in mammalian mitochondria. Solving its crystal structure revealed almost perfect alignment of the MBL domain with CPSF73, as well as to other ribonucleases of the MBL superfamily. Recombinant human LACTB2 displayed robust endoribonuclease activity on ssRNA with a preference for cleavage after purine-pyrimidine sequences. Mutational analysis identified an extended RNA-binding site. Knockdown of LACTB2 in cultured cells caused a moderate but significant accumulation of many mitochondrial transcripts, and its overexpression led to the opposite effect. Furthermore, manipulation of LACTB2 expression resulted in cellular morphological deformation and cell death. Together, this study discovered that LACTB2 is an endoribonuclease that is involved in the turnover of mitochondrial RNA, and is essential for mitochondrial function in human cells.