An interaction between an Arabidopsis poly(A) polymerase and a homologue of the 100 kDa subunit of CPSF

The Arabidopsis genome possesses a number of sequences that are predicted to encode proteins that are similar to mammalian and yeast polyadenylation factor subunits. One of these resides on chromosome V and has the potential to encode a polypeptide related to the 100 kDa subunit of the mammalian cleavage and polyadenylation specificity factor (CPSF). This gene encodes a ca. 2400 nucleotide mRNA that in turn can be translated to yield a polypeptide that is 39% identical to the mammalian CPSF100 protein. Antibodies raised against the Arabidopsis protein recognized distinctive polypeptides in nuclear extracts prepared from pea and wheat germ, consistent with the hypothesis that the Arabidopsis protein is resident in a nuclear polyadenylation complex. Interestingly, the Arabidopsis CPSF100 was found to interact with a portion of a nuclear poly(A) polymerase. This interaction was attributable to a 60 amino acid domain in the CPSF100 polypeptide and the N-terminal 220 amino acids of the poly(A) polymerase. An analogous interaction has yet to be described in other eukaryotes. The interaction with PAP thus indicates that the plant CPSF100 polypeptide is likely part of the 3-end processing machinery, but suggests that this complex may function differently in plants than it does in mammals and yeast.     

Characterization of a cDNA encoding the 55 kDa B regulatory subunit of Arabidopsis protein phosphatase 2A

Type 2A serine/threonine protein phosphatases (PP2A) are key components in the regulation of signal transduction and control of cell metabolism. The activity of these protein phosphatases is modulated by regulatory subunits. While PP2A activity has been characterized in plants, little is known about its regulation. We used the polymerase chain reaction to amplify a segment of a cDNA encoding the B regulatory subunit of PP2A from Arabidopsis. The amplified DNA fragment of 372 nucleotides was used as a probe to screen an Arabidopsis cDNA library and a full-length clone (AtB) of 2.1 kbp was isolated. The predicted protein encoded by AtB is 43 to 46% identical and 53 to 56% similar to its yeast and mammalian counterparts, and contains three unique regions of amino acid insertions not present in the animal B regulatory subunit. Genomic Southern blots indicate the Arabidopsis genome contains at least two genes encoding the B regulatory subunit. In addition, other plant species also contain DNA sequences homologous to the B regulatory subunit, indicating that regulation of PP2A activity by the 55 kDa B regulatory subunit is probably ubiquitous in plants. Northern blots indicate the AtB mRNA accumulates in all Arabidopsis tissues examined, suggesting the protein product of the AtB gene performs a basic housekeeping function in plant cells.     

The 73 kD Subunit of the cleavage and polyadenylation specificity factor (CPSF) complex affects reproductive development in Arabidopsis

The cleavage and polyadenylation specificity factor (CPSF) is an important multi-subunit component of the mRNA 3′-end processing apparatus in eukaryotes. The Arabidopsis genome contains five genes encoding CPSF homologues (AtCPSF160, AtCPSF100, AtCPSF73-I, AtCPSF73-II and AtCPSF30). These CPSF homologues interact with each other in a way that is analogous to the mammalian CPSF complex or their yeast counterparts, and also interact with the Arabidopsis poly(A) polymerase (PAP). There are two CPSF73 like proteins (AtCPSF73-I and AtCPSF73-II) that share homology with the 73 kD subunit of the mammalian CPSF complex. AtCPSF73-I appears to correspond to the functionally characterized mammalian CPSF73 and its yeast counterpart. AtCPSF73-II was identified as a novel protein with uncharacterized protein homologues in other multicellular organisms, but not in yeast. Both of the AtCPSF73 proteins are targeted in the nucleus and were found to interact with AtCPSF100. They are also essential since knockout or knockdown mutants are lethal. In addition, the expression level of AtCPSF73-I is critical for Arabidopsis development because overexpression of AtCPSF73-I is lethal. Interestingly, transgenic plants carrying an additional copy of the AtCPSF73-I gene, that is, the full-length cDNA under the control of its native promoter, appeared normal but were male sterile due to delayed anther dehiscence. In contrast, we previously demonstrated that a mutation in the AtCPSF73-II gene was detrimental to the genetic transmission of female gametes. Thus, two 73 kD subunits of the AtCPSF complex appear to have special functions during flower development. The important roles of mRNA 3′-end processing machinery in modulating plant development are discussed. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users. Gene accession numbers associated with this paper: AY140902, AY140900, AY168923, AY140901     

An Arabidopsis gene homologous to mammalian and insect genes encoding the largest proteasome subunit

A gene encoding a protein with extensive homology to the largest subunit of the multicatalytic proteinase complex (proteasome) has been identified in Arabidopsis thaliana. This gene, referred to as AtPSM30, is entirely encompassed within a previously characterized radiation-induced deletion, which may thus provide the first example of a proteasome null mutation in a higher eukaryote. However, the growth rate and fertility of Arabidopsis plants do not appear to be significantly affected by this mutation, even though disruption experiments in yeast have shown that most proteasome subunits are essential. Analysis of mRNA levels in developing seedlings and mature plants indicates that expression of AtPSM30 is differentially regulated during development and is slightly induced in response to stress, as has been observed for proteasome genes in yeast, Drosophila, and mammals. Southern blot analysis indicates that the Arabidopsis genome contains numerous sequences closely related to AtPSM30, consistent with recent reports of at least two other proteasome genes in Arabidopsis. A comparison of the deduced amino acid sequences for all proteasome genes reported to date suggests that multiple proteasome subunits evolved in eukaryotes prior to the divergence of plants and animals.GenBank accession number: M98495