A novel splice variant of human XRN2 gene is mainly expressed in blood leukocyte.

The XRN2 gene (XRN2a) is the human homologue of the Saccharomyces cerevisiae RAT1 gene, which encodes a nuclear 5'-->3' exoribonuclease, and is essential for RNA metabolism and cell viability. Xrn2p/Rat1p, product of XRN2/RAT1 gene, functions in the mRNA degradation and processing of rRNAs and small nucleolar RNAs (snoRNAs) in the nucleus. Here we describe the cloning and characterization of a novel splice variant of the human XRN2 gene (XRN2b). The 3271-bp cDNA encodes a putative protein with 907 amino acid residues, which shares high homology with mouse DHM1 protein. RT-PCR analysis showed that XRN2b was mainly expressed in blood leukocyte tissue, while XRN2a was detected in several human tissues and in human tumor tissues.     

Characterization of the XRN1 gene encoding a 5'-->3' exoribonuclease: sequence data and analysis of disparate protein and mRNA levels of gene-disrupted yeast cells.

Sequencing of the XRN1 gene of Saccharomyces cerevisiae, cloned in this laboratory as a gene encoding a 160-kDa 5'-->3' exoribonuclease (XRN1), shows that it is identical to a gene (DST2 or SEP1) encoding a DNA strand transferase and to genes involved in nuclear fusion, KEM1, and plasmid stability, RAR5. To better understand the various phenotypes associated with loss of XRN1 and the enzymatic activities associated with the protein, certain characteristics of our yeast cells lacking an active gene (xrn1) have been examined. Cells are larger (average volume is x 1.5-1.8) and have an increased doubling time (x1.9-2.1). The protein synthesis rate per cell is 80-90% that of wild-type (wt) cells, and the resultant cellular protein levels are higher. The rate of the 25S and 18S rRNA synthesis is approximately 45% that of wt cells and its cellular level is about 90% that of wt cells. Levels of protein bands resolved by one-dimensional PAGE show substantial differences. Synthesis rates observed for the same protein bands, as well as measurements of several specific mRNA levels by Northern analysis, suggest disparities in mRNA levels. Results show two to four times longer half-lives of specific short-lived mRNAs. The variations in levels of protein and RNA species found in the xrn1 cells may be the cause of some of the phenotypes found associated with gene loss.     

An essential yeast gene with homology to the exonuclease-encoding XRN1/KEM1 gene also encodes a protein with exoribonuclease activity.

An essential gene, designated HKE1/RAT1, has been isolated from the yeast Saccharomyces cerevisiae and characterized. The gene encodes a protein of 116 kDa (p116) and has significant homology to another yeast gene (XRN1/KEM1) encoding a related protein (p175) with 5'-->3' exonuclease activity as well as activities involving chromosomal DNA pairing and mechanics. Preliminary analysis of an hke1ts mutant reveals a precipitous decline in the translation of mRNA at the nonpermissive temperature. Sporulation of heterozygous HKE1/hke1::URA3 diploids reveals that this gene, unlike the highly related XRN1/KEM1 gene, is essential for cell viability. Overexpression of the homologous gene product, p175, failed to rescue cells lacking a functional p116. In vitro studies demonstrate that p116 is a protein with 5'-->3' exoribonuclease activity, a major activity of the related p175. An immunoreactive RNase activity of 116 kDa is abolished with antiserum against p116. Both the level of this protein and the RNase activity correlate with HKE1 gene dosage. The RNase activity purifies coincidentally with a previously described 116-kDa RNase having 5'-->3' exoribonuclease activity.     

Saccharomyces cerevisiae RAI1 (YGL246c) is homologous to human DOM3Z and encodes a protein that binds the nuclear exoribonuclease Rat1p

The RAT1 gene of Saccharomyces cerevisiae encodes a 5'-->3' exoribonuclease which plays an essential role in yeast RNA degradation and/or processing in the nucleus. We have cloned a previously uncharacterized gene (YGL246c) that we refer to as RAI1 (Rat1p interacting protein 1). RAI1 is homologous to Caenorhabditis elegans DOM-3 and human DOM3Z. Deletion of RAI1 confers a growth defect which can be complemented by an additional copy of RAT1 on a centromeric vector or by directing Xrn1p, the cytoplasmic homolog of Rat1p, to the nucleus through the addition of a nuclear targeting sequence. Deletion of RAI1 is synthetically lethal with the rat1-1(ts) mutation and shows genetic interaction with a deletion of SKI2 but not XRN1. Polysome analysis of an rai1 deletion mutant indicated a defect in 60S biogenesis which was nearly fully reversed by high-copy RAT1. Northern blot analysis of rRNAs revealed that rai1 is required for normal 5.8S processing. In the absence of RAI1, 5.8S(L) was the predominant form of 5.8S and there was an accumulation of 3'-extended forms but not 5'-extended species of 5. 8S. In addition, a 27S pre-rRNA species accumulated in the rai1 mutant. Thus, deletion of RAI1 affects both 5' and 3' processing reactions of 5.8S rRNA. Consistent with the in vivo data suggesting that RAI1 enhances RAT1 function, purified Rai1p stabilized the in vitro exoribonuclease activity of Rat1p.     

Identification of an exoribonuclease homolog, CaKEM1/CaXRN1, in Candida albicans and its characterization in filamentous growth

KEM1/XRN1 and RAT1 are two known exoribonuclease genes in Saccharomyces cereivsiae and encode a cytoplasmic and nuclear exoribonuclease, respectively. CaKEM1/CaXRN1 and CaRAT1, the Candida albicans homologs of 5'-->3' exoribonuclease genes, were identified by protein sequence comparisons and by functional complementation of the S. cerevisiae kem1/xrn1 null mutation. The deduced amino acid sequences of CaKEM1 and CaRAT1 show 51% and 55% identities to those of the S. cerevisiae KEM1 and RAT1, respectively. The exonuclease motifs were found to be highly conserved in CaKem1p and CaRat1p. We disrupted two chromosomal copies of CaKEM1 in a diploid C. albicans strain and demonstrate that C. albicans kem1/kem1 mutants are defective in filamentous growth on filamentous-inducing media. These results imply that CaKEM1 is involved in filamentous growth of C. albicans.     

Rat1p and Xrn1p are functionally interchangeable exoribonucleases that are restricted to and required in the nucleus and cytoplasm, respectively.

XRN1 encodes an abundant cytoplasmic exoribonuclease, Xrn1p, responsible for mRNA turnover in yeast. A screen for bypass suppressors of the inviability of xrn1 ski2 double mutants identified dominant alleles of RAT1, encoding an exoribonuclease homologous with Xrn1p. These RAT1 alleles restored XRN1-like functions, including cytoplasmic RNA turnover, wild-type sensitivity to the microtubule-destabilizing drug benomyl, and sporulation. The mutations were localized to a region of the RAT1 gene encoding a putative bipartite nuclear localization sequence (NLS). Fusions to green fluorescent protein were used to demonstrate that wild-type Rat1p is localized to the nucleus and that the mutant alleles result in mislocalization of Rat1p to the cytoplasm. Conversely, targeting Xrn1p to the nucleus by the addition of the simian virus 40 large-T-antigen NLS resulted in complementation of the temperature sensitivity of a rat1-1 strain. These results indicate that Xrn1p and Rat1p are functionally interchangeable exoribonucleases that function in and are restricted to the cytoplasm and nucleus, respectively. It is likely that the higher eukaryotic homologs of these proteins will function similarly in the cytoplasm and nucleus.     

Decoying the cap- mRNA degradation system by a double-stranded RNA virus and poly(A)- mRNA surveillance by a yeast antiviral system.

The major coat protein of the L-A double-stranded RNA virus of Saccharomyces cerevisiae covalently binds m7 GMP from 5' capped mRNAs in vitro. We show that this cap binding also occurs in vivo and that, while this activity is required for expression of viral information (killer toxin mRNA level and toxin production) in a wild-type strain, this requirement is suppressed by deletion of SKI1/XRN1/SEP1. We propose that the virus creates decapped cellular mRNAs to decoy the 5'-->3' exoribonuclease specific for cap- RNA encoded by XRN1. The SKI2 antiviral gene represses the copy numbers of the L-A and L-BC viruses and the 20S RNA replicon, apparently by specifically blocking translation of viral RNA. We show that SKI2, SKI3, and SKI8 inhibit translation of electroporated luciferase and beta-glucuronidase mRNAs in vivo, but only if they lack the 3' poly(A) structure. Thus, L-A decoys the SKI1/XRN1/SEP1 exonuclease directed at 5' uncapped ends, but translation of the L-A poly(A)- mRNA is repressed by Ski2,3,8p. The SKI2-SKI3-SKI8 system is more effective against cap+ poly(A)- mRNA, suggesting a (nonessential) role in blocking translation of fragmented cellular mRNAs.     

Yeast cells lacking 5''-->3'' exoribonuclease 1 contain mRNA species that are poly(A) deficient and partially lack the 5'' cap structure.

Analysis of the slowed turnover rates of several specific mRNA species and the higher cellular levels of some of these mRNAs in Saccharomyces cerevisiae lacking 5'-->3' exoribonuclease 1 (xrn1 cells) has led to the finding that these yeast contain higher amounts of essentially full-length mRNAs that do not bind to oligo(dT)-cellulose. On the other hand, the length of mRNA poly(A) chains found after pulse-labeling of cells lacking the exoribonuclease, the cellular rate of synthesis of oligo(dT)-bound mRNA, and the initial rate of its deadenylation appeared quite similar to the same measurements in wild-type yeast cells. Examination of the 5' cap structure status of the poly(A)-deficient mRNAs by comparative analysis of the m7G content of poly(A)- and poly(A)+ RNA fractions of wild-type and xrn1 cells suggested that the xrn1 poly(A)- mRNA fraction is low in cap structure content. Further analysis of the 5' termini by measurements of the rate of 5'-->3' exoribonuclease 1 hydrolysis of specific full-length mRNA species showed that approximately 50% of the xrn1 poly(A)-deficient mRNA species lack the cap structure. Primer extension analysis of the 5' terminus of ribosomal protein 51A (RP51A) mRNA showed that about 30% of the poly(A)-deficient molecules of the xrn1 cells are slightly shorter at the 5' end. The finding of some accumulation of poly(A)-deficient mRNA species partially lacking the cap structure together with the reduction of the rate of mRNA turnover in cells lacking the enzyme suggest a possible role for 5'-->3' exoribonuclease 1 in the mRNA turnover process.     

RNA deadenylation and decay in plants

In eukaryotic cells, RNA levels are tightly regulated in a spatio-temporal manner to maintain the protein levels necessary for cell growth, differentiation and division. To cope with developmental and rapid environmental changes, RNAs that are no longer required by the cell undergo degradation via the mRNA decay process. A number of players involved in RNA degradation pathways have been identified for the last two decades. A wealth of information about the process of mRNA deadenylation and decay in yeast and other model organisms is currently available; however, very limited information is available in plants, including Arabidopsis. Nevertheless, the efforts of various plant research groups are continuously extending our understanding of this complicated process. Here, we summarize our current knowledge of RNA decay in yeast and compare this information with the progress from Arabidopsis. This review will especially focus on the structure and function of the deadenylation complex, 5′ to 3′ exoribonuclease (XRN)-mediated decay pathways and the exosome-mediated 3′ to 5′ decay pathway in plants.