State of decay: an update on plant mRNA turnover.

Proper degradation of plant messenger RNA is crucial for the maintenance of cellular and organismal homeostasis, and it must be properly regulated to enable rapid adjustments in response to endogenous and external cues. Only a few dedicated studies have been done so far to address the fundamental mechanisms of mRNA decay in plants, especially as compared with fungal and mammalian model systems. Consequently, our systems-level understanding of plant mRNA decay remains fairly rudimentary. Nevertheless, a number of serendipitous findings in recent years have reasserted the central position of the regulated mRNA decay in plant physiology. In addition, the meteoric rise to prominence of the plant small RNA field has spawned a renewed interest in the general plant mRNA turnover pathways. Combined with the advent of widely accessible microarray platforms, these advances allow for a renewed hope of rapid progress in our understanding of the fundamental rules governing regulated mRNA degradation in plants. This chapter summarizes recent findings in this field.

Analysis of an essential requirement for the poly(A) binding protein function using cross-species complementation.

Poly(A) binding protein (PABP) is an essential, well-conserved, multifunctional protein involved in translational initiation, mRNA biogenesis, and degradation [1--5]. We have used a cross-species complementation approach to address the nature of the essential requirement for PABP in yeast. The expression of Pab3p, a member of the Arabidopsis thaliana PABP multigene family, rescues the lethal phenotype associated with the loss of the yeast Pab1p. However, Pab3p neither protects the mRNA 5' cap from premature removal, nor does it support poly(A)-dependent translational initiation or the synergistic enhancement of translation by the poly(A) tail and 5' cap in yeast. However, Pab3p corrects the temporal lag prior to the entry of the mRNA into the degradation pathway characteristic of pab1 Delta yeast strains. Furthermore, this lag correction by Pab3p requires Pan3p, a subunit of poly(A) nuclease, an enzyme involved in the mRNA 3'-end processing. Importantly, the substitution of Pab3p for the yeast Pab1p is synthetically lethal with the PAN3 gene deletion. These results show that the function of PABP in mRNA biogenesis alone could be sufficient to support cell viability in yeast.

Characterization of relic DNA from barley genome.

High-molecular-weight "relic" DNA fraction can be electrophoretically separated from the bulk of barley DNA digested with different restriction enzymes. We have cloned and analyzed a population of relic DNA fragments. The majority of AluI-relic DNA clones contained barley simple sequence satellite DNA and other families of repetitive DNA. One of these families, designated HvRT, has been analyzed in detail. This family is composed of tandemly arranged 118-bp monomers and is present in 7 × 10(5) copies in the barley genome. Clones representing the HvRT family were sequenced. HvRT repeats were found to contain high levels of methylated cytosine. The HvRT family was found in the genomes of H. vulgare, H. leporinum, H. murinum, H. jubatum, but not in H. marinum, H. geniculatum, and wheat. Different barley species and cultivars show restriction fragment length polymorphism with the HvRT probe. Chromosome-specific subfamilies of HvRT were found to be present on different barley chromosomes, providing the possibility of using the HvRT probe as a chromosome specific marker. HvRT fragments up to 810 kbp in length were resolved by pulsed field gel electrophoresis.

Differential organ-specific expression of three poly(A)-binding-protein genes from Arabidopsis thaliana.

Poly(A)-binding protein (PABP) is considered an essential component of a eukaryotic cell; deletion of the PABP-coding gene in yeast leads to a lethal phenotype. PABP is implicated in numerous aspects of posttranscriptional regulation, including mRNA turnover and translational initiation. A nested set of degenerate PCR primers designed from regions conserved among yeast, Xenopus, and human PABP sequences was used to amplify genomic DNA fragments from Arabidopsis thaliana. Hybridization screening of genomic and cDNA libraries with a genomic PCR probe led to the isolation of three diverse Arabidopsis genes encoding PABPs, PAB1, PAB3, and PAB5. All three sequences contain the expected four RNA-recognition motifs. Sequence diversity between these genes equals or exceeds the diversity among animal and fungal sequences. One of the genes, PAB5, and its cDNA were completely sequenced. Its open reading frame encodes a 73.2-kDa protein containing a number of amino acid motifs characteristic of PABPs from different species. Moreover, in vitro synthesized PAB5 protein bound to poly(A)-Sepharose with high specificity. All three genes isolated showed organ-specific patterns of expression. PAB5 and PAB3 RNAs were detected only in floral organs, with the highest level of expression in immature flowers. PAB1 RNA was observed predominantly in roots, was less abundant in immature flowers, and was not detected in any other organ examined (stems, leaves, mature flowers, siliques). This suggests a potentially unique role for PABPs in organ-specific posttranscriptional regulation in plants.

A pollen-, ovule-, and early embryo-specific poly(A) binding protein from Arabidopsis complements essential functions in yeast.

Poly(A) tails of eukaryotic mRNAs serve as targets for regulatory proteins affecting mRNA stability and translation. Differential mRNA polyadenylation and deadenylation during gametogenesis and early development are now widely recognized as mechanisms of translational regulation in animals, but they have not been observed in plants. Here, we report that the expression of the PAB5 gene encoding one of the poly(A) binding proteins (PABPs) in Arabidopsis is restricted to pollen and ovule development and early embryogenesis. Furthermore, PAB5 is capable of rescuing a PABP-deficient yeast strain by partially restoring both poly(A) shortening and translational initiation functions of PABP. However, PAB5 did not restore the linkage of deadenylation and decapping, thus demonstrating that this function of PABP is not essential for viability. Also, like endogenous PABP, PAB5 expressed in yeast demonstrated genetic interaction with a recently characterized yeast protein SIS1, which is also involved in translational initiation. We propose that PAB5 encodes a post-transcriptional regulatory factor acting through molecular mechanisms similar to those reported for yeast PABP. This factor may have evolved further to post-transcriptionally regulate plant sexual reproduction and early development.

Arabidopsis thaliana poly (A) binding protein 2 (PAB2) functions in yeast translational and mRNA decay processes.

The single yeast gene (PAB1) encoding poly (A) binding protein (PABP) has several roles in post-transcriptional processes, including translation initiation and mRNA decay. PABP is encoded by a large gene family in plants. Within Arabidopsis thaliana, the several characterized PABP genes exhibit an extreme degree of sequence divergence and are differentially expressed. Arabidopsis PAB2 is expressed in distinct tissues or during defined developmental windows in most plant organs. In this study we demonstrate that PAB2 restores viability to a yeast pab1 mutant strain. Yeast strains containing wild-type, null (PAB2s) and temperature sensitive (PAB2ts) alleles of PAB2 were used to explore the molecular functions of the plant protein. PAB2 can participate in poly (A) tail shortening, thus demonstrating that it interacts with the yeast poly(A) nuclease complex. PAB2 is required for translation, helping to maintain intact polysome structures. Consistent with its role in translation initiation, poly (A) was found to enhance PAB2 binding to Arabidopsis eIF-iso4G in vitro. In addition, PAB2 can partially restore the linkage between deadenylation, decapping and mRNA decay in yeast. Taken together, our results suggest that Arabidopsis PAB2 participates in many of the same complex post-transcriptional processes identified for yeast PAB1, and is functionally distinct from other characterized Arabidopsis PABPs.

Conserved expression of Arabidopsis thaliana poly (A) binding protein 2 (PAB2) in distinct vegetative and reproductive tissues.

The poly(A) tails of eukaryotic mRNAs are complexed with poly(A) binding protein (PABP). The poly(A)-PABP complex is central to the efficient translation initiation and control of poly (A) tail length and is required in some pathways of mRNA decay. A large gene family encodes PABPs in Arabidopsis thaliana. In striking contrast to the floral and root specific expression of three previously reported Arabidopsis PABPs, we demonstrate that RNA and protein for one highly diverse member of this family, PAB2, are expressed in roots, stems, leaves, flowers, pollen and siliques of Arabidopsis. However, cell-type specific analysis of a PAB2 reporter gene fusion revealed that PAB2 is spatially and temporally regulated in each organ. For example, strong expression was detected only in the stele and meristem region of roots and a dramatic decrease in expression was observed upon fertilization of ovules. Furthermore, the PAB2-reporter construct gave a nearly identical expression pattern in transgenic tobacco, demonstrating that PAB2 expression is under strong selective constraint. The PAB2-reporter was also strongly expressed in the transmittal tissues of both Arabidopsis and tobacco, raising the possibility of its involvement in the pollination-dependent poly(A) tail shortening of transmittal tissue specific mRNAs previously reported in tobacco (Wang et al. 1996, Plant J. 9, 715-727). In view of its potential role in poly(A) tail shortening, we demonstrated the strong and distinct presence of PAB2 protein in transmittal tissues of Arabidopsis. The evolutionary and functional implications of the expression pattern of PAB2 and its possible functional roles in post-transcriptional regulation in transmittal tissues are discussed.

Poly(A) tail-dependent exonuclease AtRrp41p from Arabidopsis thaliana rescues 5.8 S rRNA processing and mRNA decay defects of the yeast ski6 mutant and is found in an exosome-sized complex in plant and yeast cells.

Eukaryotic 3'-->5' exonucleolytic activities are essential for a wide variety of reactions of RNA maturation and metabolism, including processing of rRNA, small nuclear RNA, and small nucleolar RNA, and mRNA decay. Two related but distinct forms of a complex containing 10 3'-->5' exonucleases, the exosome, are found in yeast nucleus and cytoplasm, respectively, and related complexes exist in human cells. Here we report on the characterization of the AtRrp41p, an Arabidopsis thaliana homolog of the Saccharomyces cerevisiae exosome subunit Rrp41p (Ski6p). Purified recombinant AtRrp41p displays a processive phosphorolytic exonuclease activity and requires a single-stranded poly(A) tail on a substrate RNA as a "loading pad." The expression of the Arabidopsis RRP41 cDNA in yeast rescues the 5.8 S rRNA processing and 3'-->5' mRNA degradation defects of the yeast ski6-100 mutant. However, neither of these defects can explain the conditional lethal phenotype of the ski6-100 strain. Importantly, AtRrp41p shares additional function(s) with the yeast Rrp41p which are essential for cell viability because it also rescues the rrp41 (ski6) null mutant. AtRrp41p is found predominantly in a high molecular mass complex in Arabidopsis and in yeast cells, and it interacts in vitro with the yeast Rrp44p and Rrp4p exosome subunits, suggesting that it can participate in evolutionarily conserved interactions that could be essential for the integrity of the exosome complex.

Sequence-based identification of T-DNA insertion mutations in Arabidopsis: actin mutants act2-1 and act4-1.

A method is presented to facilitate the isolation of mutations in plant genes, which requires knowledge of the target gene or protein sequence, and is independent of mutant phenotype. The polymerase chain reaction was used to amplify the junctions between a T-DNA insert and the gene of interest from pools of mutant plant lines. The approach was used to identify mutations in Arabidopsis thaliana actin genes. The Arabidopsis genome encodes 10 actins in six ancient subclasses each with distinct expression patterns. Primers in the T-DNA border and highly degenerate actin primers, designed from conserved amino acid motifs, were used to prime the amplification. The PCR products were transferred to filters and probed for actin at low stringency. Thus, mutations in all 10 actin genes were screened for simultaneously. Mutations in the vegetative constitutive actin gene, ACT2, and the pollen-specific actin gene, ACT4, were identified in a population of 5300 lines containing approximately 1.5 T-DNA insertions per line. The screen was sensitive enough that actin insertion alleles were easily distinguished among pools of 100 plant lines. PCR techniques were used which accelerated the purification of mutant lines, and segregation, physical mapping, and sequencing of the act2-1 and act4-1 mutations. This strategy should be generally useful in screening mutant libraries made with a variety of plant insertion elements for mutations in any known sequence.