Adenosine-rich elements present in the 5'-untranslated region of PABP mRNA can selectively reduce the abundance and translation of CAT mRNAs in vivo.

The poly(A)-binding protein (PABP) is a highly conserved eukaryotic protein whose synthesis is regulated at the post-transcriptional level. The binding of PABP to the poly(A)-rich element found in the 5'-untranslated region (5'UTR) of PABP mRNA specifically inhibits its own translation. In this report, we show that similar adenosine-rich elements in the 5'UTR of the chloramphenicol acetyl-transferase (CAT) gene can significantly reduce the reporter mRNA abundance and translation in human 293 cells. The reduction in mRNA level, but not CAT expression, is dependent on the size of the 5'UTR poly(A) element. Furthermore, one 5'UTR-tethered PABP molecule is enough to inhibit CAT expression without affecting its mRNA level. We propose that the control of PABP synthesis may involve mRNA decay and the repression of translation.

Ectopic expression of a Meloidogyne incognita dorsal gland protein in tobacco accelerates the formation of the nematode feeding site.

Meloidogyne spp., plant-parasitic nematodes present worldwide, are intensively studied because of the damage caused to a large variety of agronomically important crops. Several reports indicate that proteins from the Meloidogyne spp. dorsal gland might play an important role to allow proper establishment of a functional nematode feeding site. The precise role of these proteins in the process of feeding cell development is unknown. To gain insights into the function of these secreted M. incognita proteins, we constitutively (ectopically) expressed the nematodes dorsal gland protein 7E12 in tobacco plants. It was found that the number of galls at 8 and 16 days after nematode infection was significantly higher in transgenic plants compared to control plants. Eggs from nematodes in transgenic plants hatched faster than those in control plants. Histological analysis of nematode induced galls in transgenic plants clearly shows a different morphology. Giant feeding cells harbor more vacuoles and an increased amount of cell wall invaginations, while neighboring cells surrounding feeding cells are more numerous. These results suggest that the presence of the 7E12 protein in tobacco accelerates gall formation. This assumption is supported by our data illustrating faster gall formation and egg eclosion in transgenic plants.

Protective effects of a cysteine proteinase propeptide expressed in transgenic soybean roots.

Sedentary endoparasitic nematodes cause extensive damage to a large number of ornamental plants and food crops, with estimated economical losses over 100 billion US$ worldwide. Various efforts have put forth in order to minimize nematode damage, which typically involve the use of nematicides that have high cost and enhanced toxicity to humans and the environment. Additionally, different strategies have been applied in order to develop genetically modified plants with improved nematode resistance. Among the strategies are anti-invasion and migration, feeding-cell attenuation, and anti-nematode feeding. In the present study, we focus on anti-nematode feeding, which involves the evaluation and potential use of the cysteine proteinase (CPs) propeptide as a control alternative. The cysteine proteinase prodomain, isolated from Heterodera glycines (HGCP prodomain), is a natural inhibitory peptide used to transform soybean cotyledons using Agrobacterium rhizogenes. Genetically modified soybean roots expressing the propeptide were detected by Western blot and expression levels were measured by ELISA (around 0.3%). The transgenic roots expressing the propeptide were inoculated with a thousand H. glycines at the second juvenile stage, and a remarkable reduction in the number of females and eggs was observed. A reduction of female length and diameter was also observed after 35 days post-inoculation. Furthermore, the H. glycines mature protein was detected in females fed on soybean transformed root expressing or not expressing the propeptide. The data presented here indicate that the HGCP propeptide can reduce soybean cyst nematode infection and this strategy could be applied in the near future to generate resistant crop cultivars.

Identification of a C-terminal poly(A)-binding protein (PABP)-PABP interaction domain: role in cooperative binding to poly (A) and efficient cap distal translational repression.

The poly(A)-binding protein (PABP), bound to the 3' poly(A) tail of eukaryotic mRNAs, plays critical roles in mRNA translation and stability. PABP autoregulates its synthesis by binding to a conserved A-rich sequence present in the 5'-untranslated region of PABP mRNA and repressing its translation. PABP is composed of two parts: the highly conserved N terminus, containing 4 RNA recognition motifs (RRMs) responsible for poly(A) and eIF4G binding; and the more variable C terminus, which includes the recently described PABC domain, and promotes intermolecular interaction between PABP molecules as well as cooperative binding to poly(A). Here we show that, in vitro, GST-PABP represses the translation of reporter mRNAs containing 20 or more A residues in their 5'-untranslated regions and remains effective as a repressor when an A61 tract is placed at different distances from the cap, up to 126 nucleotides. Deletion of the PABP C terminus, but not the PABC domain alone, significantly reduces its ability to inhibit translation when bound to sequences distal to the cap, but not to proximal ones. Moreover, cooperative binding by multiple PABP molecules to poly(A) requires the C terminus, but not the PABC domain. Further analysis using pull-down assays shows that the interaction between PABP molecules, mediated by the C terminus, does not require the PABC domain and is enhanced by the presence of RRM 4. In vivo, fusion proteins containing parts of the PABP C terminus fused to the viral coat protein MS2 have an enhanced ability to prevent the expression of chloramphenicol acetyltransferase reporter mRNAs containing the MS2 binding site at distal distances from the cap. Altogether, our results identify a proline- and glutamine-rich linker located between the RRMs and the PABC domain as being strictly required for PABP/PABP interaction, cooperative binding to poly(A) and enhanced translational repression of reporter mRNAs in vitro and in vivo.