Polyadenylated versions of small non-coding RNAs in Saccharomyces cerevisiae are degraded by Rrp6p/Rrp47p independent of the core nuclear exosome.

In Saccharomyces cerevisiae, polyadenylated forms of mature (and not precursor) small non-coding RNAs (sncRNAs) those fail to undergo proper 3'-end maturation are subject to an active degradation by Rrp6p and Rrp47p, which does not require the involvement of core exosome and TRAMP components. In agreement with this finding, Rrp6p/Rrp47p is demonstrated to exist as an exosome-independent complex, which preferentially associates with mature polyadenylated forms of these sncRNAs. Consistent with this observation, a C-terminally truncated version of Rrp6p (Rrp6p-ΔC2) lacking physical association with the core nuclear exosome supports their decay just like its full-length version. Polyadenylation is catalyzed by both the canonical and non-canonical poly(A) polymerases, Pap1p and Trf4p. Analysis of the polyadenylation profiles in WT and rrp6-Δ strains revealed that the majority of the polyadenylation sites correspond to either one to three nucleotides upstream or downstream of their mature ends and their poly(A) tails ranges from 10-15 adenylate residues. Most interestingly, the accumulated polyadenylated snRNAs are functional in the rrp6-Δ strain and are assembled into spliceosomes. Thus, Rrp6p-Rrp47p defines a core nuclear exosome-independent novel RNA turnover system in baker's yeast targeting imperfectly processed polyadenylated sncRNAs that accumulate in the absence of Rrp6p.

RRM1 and PAB domains of translation initiation factor eIF4G (Tif4631p) play a crucial role in the nuclear degradation of export-defective mRNAs in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the CBC-Tif4631p-dependent exosomal targeting (CTEXT) complex consisting of Cbc1/2p, Tif4631p and Upf3p promotes the exosomal degradation of aberrantly long 3'-extended, export-defective transcripts and a small group of normal (termed 'special') mRNAs. We carried out a systematic analysis of all previously characterized functional domains of the major CTEXT component Tif4631p by deleting each of them and interrogating their involvement in the nuclear surveillance of abnormally long 3'-extended and export-defective messages. Our analyses show that the N-terminal RNA recognition motif 1 (RRM1) and poly(A)-binding protein (PAB) domains of Tif4631p, spanning amino acid residues, 1-82 and 188-299 in its primary structure, respectively, play a crucial role in degrading these aberrant messages. Furthermore, the physical association of the nuclear exosome with the altered/variant CTEXT complex harboring any of the mutant Tif4631p proteins lacking either the RRM1 or PAB domain becomes abolished. This finding indicates that the association between CTEXT and the exosome is accomplished via interaction between these Tif4631p domains with the major exosome component, Rrp6p. Abolition of interaction between altered CTEXT (harboring any of the RRM1/PAB-deleted versions of Tif4631p) and the exosome further leads to the impaired recruitment of the RNA targets to the Rrp6p subunit of the exosome carried out by the RRM1/PAB domains of Tif4631p. When analyzing the Tif4631p-interacting proteins, we identified a DEAD-box RNA helicase (Dbp2p), as an interacting partner that turned out to be a previously unknown component of CTEXT. The present study provides a more complete description of the CTEXT complex and offers insight into the functional relationship of this complex with the nuclear exosome.

The Sequential Recruitments of Rab-GTPase Ypt1p and the NNS Complex onto pre-HAC1 mRNA Promote Its Nuclear Degradation in Baker's Yeast.

Induction of unfolded protein response involves activation of transcription factor Hac1p that is encoded by HAC1 pre-mRNA harboring an intron and a bipartite element (BE), which is subjected to nuclear mRNA decay by the nuclear exosome/Cbc1p-Tif4631p-dependent Exosome Targeting (CTEXT) complex. Using a combination of genetic and biochemical approaches, we demonstrate that a Rab-GTPase Ypt1p controls unfolded protein response signaling dynamics. This regulation relies on the nuclear localization of a small fraction of the cellular Ypt1p pool in the absence of endoplasmic reticulum (ER)-stress causing a strong association of the nuclear Ypt1p with pre-HAC1 mRNA that eventually promotes sequential recruitments of NNS, CTEXT, and the nuclear exosome onto this pre-mRNA. Recruitment of these decay factors onto pre-HAC1 mRNA is accompanied by its rapid nuclear decay that produces a precursor RNA pool lacking functional BE thereby causing its inefficient targeting to Ire1p foci leading to their diminished splicing and translation. ER stress triggers rapid relocalization of the nuclear pool of Ypt1p to the cytoplasm leading to its dissociation from pre-HAC1 mRNA thereby causing decreased recruitment of these decay factors to precursor HAC1 RNA leading to its diminished degradation. Reduced decay results in an increased abundance of pre-HAC1 mRNA with intact functional BE leading to its enhanced recruitment to Ire1p foci.

Determination of the Stability and Intracellular (Intra-Nuclear) Targeting and Recruitment of Pre-HAC1 mRNA in the Saccharomyces cerevisiae During the Activation of UPR.

Nuclear degradation of pre-HAC1 mRNA and its subsequent targeting plays a vital role in the activation as well as attenuation of Unfolded Protein Response (UPR) in Saccharomyces cerevisiae. Accurate measurement of the degradation of precursor HAC1 mRNA therefore appears vital to determine the phase of activation or attenuation of this important intracellular signaling pathway. Typically, pre-HAC1 mRNA degradation is measured by the transcription shut-off experiment in which RNA Polymerase II transcription is inhibited by a potent transcription inhibitor to prevent the de novo synthesis of all Polymerase II transcripts followed by the measurement of the steady-state levels of a specific (e.g., pre-HAC1) mRNA at different times after the inhibition of the transcription. The rate of the decay is subsequently determined from the slope of the decay curve and is expressed as half-life (T1/2). Estimation of the half-life values and comparison of this parameter determined under different physiological cues (such as in absence or presence of redox/ER/heat stress) gives a good estimate of the stability of the mRNA under these conditions and helps gaining an insight into the mechanism of the biological process such as activation or attenuation of UPR.Intra-nuclear targeting of the pre-HAC1 mRNA from the site of its transcription to the site of non-canonical splicing, where the kinase-endonuclease Ire1p clusters into the oligomeric structures constitutes an important aspect of the activation of Unfolded Protein Response pathway. These oligomeric structures are detectable as the Ire1p foci/spot in distinct locations across the nuclear-ER membrane under confocal micrograph using immunofluorescence procedure. Extent of the targeting of the pre-HAC1 mRNA is measurable in a quantified manner by co-expressing fluorescent-labeled pre-HAC1 mRNA and Ire1p protein followed by estimating their co-localization using FACS (Fluorescence-Activated Cell Sorter) analysis. Here, we describe detailed protocol of both determination of intra-nuclear decay rate and targeting-frequency of pre-HAC1 mRNA that were optimized in our laboratory.

Nrd1p identifies aberrant and natural exosomal target messages during the nuclear mRNA surveillance in Saccharomyces cerevisiae.

Nuclear degradation of aberrant mRNAs in Saccharomyces cerevisiae is accomplished by the nuclear exosome and its cofactors TRAMP/CTEXT. Evidence from this investigation establishes a universal role of the Nrd1p-Nab3p-Sen1p (NNS) complex in the nuclear decay of all categories of aberrant mRNAs. In agreement with this, both nrd1-1 and nrd1-2 mutations impaired the decay of all classes of aberrant messages. This phenotype is similar to that displayed by GAL::RRP41 and rrp6-Δ mutant yeast strains. Remarkably, however, nrd1ΔCID mutation (lacking the C-terminal domain required for interaction of Nrd1p with RNAPII) only diminished the decay of aberrant messages with defects occurring during the early stage of mRNP biogenesis, without affecting other messages with defects generated later in the process. Co-transcriptional recruitment of Nrd1p on the aberrant mRNAs was vital for their concomitant decay. Strikingly, this recruitment on to mRNAs defective in the early phases of biogenesis is solely dependent upon RNAPII. In contrast, Nrd1p recruitment onto export-defective transcripts with defects occurring in the later stage of biogenesis is independent of RNAPII and dependent on the CF1A component, Pcf11p, which explains the observed characteristic phenotype of nrd1ΔCID mutation. Consistently, pcf11-2 mutation displayed a selective impairment in the degradation of only the export-defective messages.

Localization elements and zip codes in the intracellular transport and localization of messenger RNAs in Saccharomyces cerevisiae.

Intracellular trafficking and localization of mRNAs provide a mechanism of regulation of expression of genes with excellent spatial control. mRNA localization followed by localized translation appears to be a mechanism of targeted protein sorting to a specific cell-compartment, which is linked to the establishment of cell polarity, cell asymmetry, embryonic axis determination, and neuronal plasticity in metazoans. However, the complexity of the mechanism and the components of mRNA localization in higher organisms prompted the use of the unicellular organism Saccharomyces cerevisiae as a simplified model organism to study this vital process. Current knowledge indicates that a variety of mRNAs are asymmetrically and selectively localized to the tip of the bud of the daughter cells, to the vicinity of endoplasmic reticulum, mitochondria, and nucleus in this organism, which are connected to diverse cellular processes. Interestingly, specific cis-acting RNA localization elements (LEs) or RNA zip codes play a crucial role in the localization and trafficking of these localized mRNAs by providing critical binding sites for the specific RNA-binding proteins (RBPs). In this review, we present a comprehensive account of mRNA localization in S. cerevisiae, various types of localization elements influencing the mRNA localization, and the RBPs, which bind to these LEs to implement a number of vital physiological processes. Finally, we emphasize the significance of this process by highlighting their connection to several neuropathological disorders and cancers. This article is categorized under: RNA Export and Localization > RNA Localization.

A Nuclear Zip Code in SKS1 mRNA Promotes Its Slow Export, Nuclear Retention, and Degradation by the Nuclear Exosome/DRN in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, a special class of mRNAs representing a subset of otherwise normal transcripts displays very slow export and an unusually long intra-nuclear dwell time. This prolonged nuclear retention leads to their rapid degradation in the nucleus by the nuclear exosome and DRN (Decay of RNA in the Nucleus) apparatus. We previously attributed their slow export to one or more hypothetical cis-acting, export-retarding element(s). Here, we identified such a cis-element (hereafter referred to as "nuclear zip code") in SKS1 mRNA, a representative of this class of transcripts. Deletion analysis of SKS1 mRNA identified a 202-nt RNA segment within the SKS1 ORF, which harbors the nuclear zip code. Removal of this segment (i) abolished slow export of the transcripts, as revealed by in situ confocal microscopy-based localization experiments, and (ii) abrogated the susceptibility of the transcripts to degradation by the nuclear exosome/DRN. Remarkably, fusing the SKS1 mRNA 202-nt nuclear zip code to the 5'-segment of CYC1 mRNA resulted in inefficient export, and susceptibility of the chimeric transcript to the nuclear exosome/DRN. These findings identify a cis-acting zip code element that is necessary and sufficient to impede nuclear export and results in its preferential nuclear retention, thereby impacting its abundance and cellular repertoire. We conclude that this element posttranscriptionally regulates SKS1 gene expression levels.

Acidic domain of WRNp is critical for autophagy and up-regulates age associated proteins.

Impaired autophagy may be associated with normal and pathological aging. Here we explore a link between autophagy and domain function of Werner protein (WRNp). Werner (WRN) mutant cell lines AG11395, AG05229 and normal aged fibroblast AG13129 display a deficient response to tunicamycin mediated endoplasmic reticulum (ER) stress induced autophagy compared to clinically unaffected GM00637 and normal young fibroblast GM03440. Cellular endoplasmic reticulum (ER) stress mediated autophagy in WS and normal aged cells is restored after transfection with wild type full length WRN, but deletion of the acidic domain from wild type WRN fails to restore autophagy. The acidic domain of WRNp was shown to regulate its transcriptional activity, and here, we show that it affects the transcription of certain proteins involved in autophagy and aging. Furthermore, siRNA mediated silencing of WRN in normal fibroblast WI-38 resulted in decrease of age related proteins Lamin A/C and Mre11.

Nuclear mRNA Surveillance Mechanisms: Function and Links to Human Disease.

Production of export-competent mRNAs involves transcription and a series of dynamic processing and modification events of pre-messenger RNAs in the nucleus. Mutations in the genes encoding the transcription and mRNP processing machinery and the complexities involved in the biogenesis events lead to the formation of aberrant messages. These faulty transcripts are promptly eliminated by the nuclear RNA exosome and its cofactors to safeguard the cells and organisms from genetic catastrophe. Mutations in the components of the core nuclear exosome and its cofactors lead to the tissue-specific dysfunction of exosomal activities, which are linked to diverse human diseases and disorders. In this article, we examine the structure and function of both the yeast and human RNA exosome complex and its cofactors, discuss the nature of the various altered amino acid residues implicated in these diseases with the speculative mechanisms of the mutation-induced disorders and project the frontier and prospective avenues of the future research in this field.

Nuclear mRNA degradation tunes the gain of the unfolded protein response in Saccharomyces cerevisiae.

Unfolded protein response (UPR) is triggered by the accumulation of unfolded proteins in the endoplasmic reticulum (ER), which is accomplished by a dramatic induction of genes encoding ER chaperones. Activation of these genes involves their rapid transcription by Hac1p, encoded by the HAC1 precursor transcript harboring an intron and a bipartite element (3'-BE) in the 3'-UTR. ER stress facilitates intracellular targeting and recruitment of HAC1 pre-mRNA to Ire1p foci (requiring 3'-BE), leading to its non-spliceosomal splicing mediated by Ire1p/Rlg1p. A critical concentration of the pre-HAC1 harboring a functional 3'-BE element is governed by its 3'→5' decay by the nuclear exosome/DRN. In the absence of stress, pre-HAC1 mRNA undergoes a rapid and kinetic 3'→5' decay leading to a precursor pool, the majority of which lack the BE element. Stress, in contrast, causes a diminished decay, thus resulting in the production of a population with an increased abundance of pre-HAC1 mRNA carrying an intact BE, which facilitates its more efficient recruitment to Ire1p foci. This mechanism plays a crucial role in the timely activation of UPR and its prompt attenuation following the accomplishment of homeostasis. Thus, a kinetic mRNA decay provides a novel paradigm for mRNA targeting and regulation of gene expression.

The interplay between transcription and mRNA degradation in Saccharomyces cerevisiae.

The cellular transcriptome is shaped by both the rates of mRNA synthesis in the nucleus and mRNA degradation in the cytoplasm under a specified condition. The last decade witnessed an exciting development in the field of post-transcriptional regulation of gene expression which underscored a strong functional coupling between the transcription and mRNA degradation. The functional integration is principally mediated by a group of specialized promoters and transcription factors that govern the stability of their cognate transcripts by "marking" them with a specific factor termed "coordinator." The "mark" carried by the message is later decoded in the cytoplasm which involves the stimulation of one or more mRNA-decay factors, either directly by the "coordinator" itself or in an indirect manner. Activation of the decay factor(s), in turn, leads to the alteration of the stability of the marked message in a selective fashion. Thus, the integration between mRNA synthesis and decay plays a potentially significant role to shape appropriate gene expression profiles during cell cycle progression, cell division, cellular differentiation and proliferation, stress, immune and inflammatory responses, and may enhance the rate of biological evolution.

DRN and TRAMP degrade specific and overlapping aberrant mRNAs formed at various stages of mRNP biogenesis in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, nuclear exosome along with TRAMP and DRN selectively eliminates diverse aberrant messages. These decay apparatuses appear to operate as independent mechanisms in the nucleus. Here, using genetic and molecular approach we systematically investigate the functional relationship between exosome, TRAMP and DRN mechanisms by examining their relative contributions in the degradation of diverse classes of aberrant nuclear mRNAs generated at various phases of mRNP biogenesis. Our findings suggest that nuclear exosome in association with the TRAMP complex exclusively degrades the transcription assembly-defective mRNPs and splice-defective intron-containing pre-mRNAs, whereas nuclear exosome along with DRN solely degrades the export-defective messages. The degradation of aberrant read-through transcripts with 3'-extensions, in contrast, requires the activity of TRAMP and DRN together along with nuclear exosome function. Thus, the profile of substrate specificity of these nuclear decay machines reflects dependency of the nuclear exosome for either TRAMP or DRN function to degrade distinct nuclear mRNAs. We propose that DRN apparatus may act as a novel ancillary factor required for the nuclear exosome function to degrade specific classes of aberrant messages.

eIF4G-an integrator of mRNA metabolism?

The eukaryotic translation initiation factor, eIF4G, plays a key functional role in the initiation of cap-dependent translation by acting as an adapter to nucleate the assembly of eIF4F complex. Together with poly(A)-binding protein and eIF3, eIF4F subsequently triggers the recruitment of 43S ribosomal pre-initiation complex to the messenger RNA template. Since eukaryotes primarily regulate translation at the level of initiation, eIF4G is implicated in the control of eukaryotic gene expression. Remarkably, emerging evidence in Saccharomyces cerevisiae indicates that eIF4G also plays a key role in nuclear mRNA biogenesis and surveillance-a finding that is in agreement with its nuclear distribution. Here, we focus on the functional involvement of eIF4G in the nucleus in modulating pre-mRNA splicing, mRNA surveillance and possibly in much-debated nuclear translation. Notably, the nature of the biochemical role of this protein in the major events of cellular mRNA metabolism emphasizes that this crucial protein factor may serve as a general integrator of mRNA functional states by acting as an adapter molecule.

N6-methyladenosine modification in mRNA: machinery, function and implications for health and diseases.

N6-methyladenosine (m(6) A) modification in mRNA is extremely widespread, and functionally modulates the eukaryotic transcriptome to influence mRNA splicing, export, localization, translation, and stability. Methylated adenines are present in a large subset of mRNAs and long noncoding RNAs (lncRNAs). Methylation is reversible, and this is accomplished by the orchestrated action of highly conserved methyltransferase (m(6) A writer) and demethylase (m(6) A eraser) enzymes to shape the cellular 'epitranscriptome'. The engraved 'methyl code' is subsequently decoded and executed by a group of m(6) A reader/effector components, which, in turn, govern the fate of the modified transcripts, thereby dictating their potential for translation. Reversible mRNA methylation thus adds another layer of regulation at the post-transcriptional level in the gene expression programme of eukaryotes that finely sculpts a highly dynamic proteome in order to respond to diverse cues during cellular differentiation, immune tolerance, and neuronal signalling.

Cbc2p, Upf3p and eIF4G are components of the DRN (Degradation of mRNA in the Nucleus) in Saccharomyces cerevisiae.

Messenger RNAs retained in the nucleus of Saccharomyces cerevisiae are subjected to a degradation system designated DRN (Degradation of mRNA in the Nucleus) that is dependent on the nuclear mRNA cap-binding protein, Cbc1p, as well as nuclear exosome component Rrp6p, a 3' to 5' exoribonuclease. DRN has been shown to act on RNAs preferentially retained in the nucleus, such as: (1) global mRNAs in export defective nup116-Δ mutant strains at the restrictive temperature; (2) a certain class of normal mRNAs called special mRNAs (e.g. IMP3 and YLR194c mRNAs); and (3) mutant mRNAs for example, lys2-187 and cyc1-512. In this study, we further identify three novel components of DRN (Cbc2p, Upf3p and Tif4631p) by employing a genetic screen and by considering proteins/factors that interact with Cbc1p. Participation of these components in DRN was confirmed by demonstrating that null alleles of these genes resulted in stabilization of the rapid decay of global mRNAs in the export defective nup116-Δ strain and of representative special mRNAs. Depletion of Tif4632p, an isoform of Tif4631p, also exhibited a partial impairment of DRN function and is therefore also considered to play a functional role in DRN. These findings clearly establish that CBC2, UPF3, and TIF4631/32 gene products participate in DRN function.

mRNA quality control pathways in Saccharomyces cerevisiae.

Efficient production of translation-competent mRNAs involves processing and modification events both in the nucleus and cytoplasm which require a number of complex machineries at both co-transcriptional and posttranscriptional levels. Mutations in the genomic sequence sometimes result in the formation of mutant nonfunctional defective messages. In addition, the enormous amounts of complexities involved in the biogenesis of mRNPs in the nucleus very often leads to the formation of aberrant and faulty messages along with their functional counterpart. Subsequent translation of these mutant and defective populations of messenger RNAs could possibly result in the unfaithful transmission of genetic information and thus is considered a threat to the survival of the cell. To prevent this possibility, mRNA quality control systems have evolved both in the nucleus and cytoplasm in eukaryotes to scrutinize various stages of mRNP biogenesis and translation. In this review, we will focus on the physiological role of some of these mRNA quality control systems in the simplest model eukaryote Saccharomyces cerevisiae.

The sld genetic defect: two intronic CA repeats promote insertion of the subsequent intron and mRNA decay.

The autosomal recessive mutation, sld, attenuates mucous cell expression in murine sublingual glands with corresponding effects on mucin 19 (Muc19). We conducted a systematic study including genetic mapping, sequencing, and functional analyses to elucidate a mutation to explain the sld phenotype in neonatal mice. Genetic mapping and gene expression analyses localized the sld mutation within the gene Muc19/Smgc, specifically attenuating Muc19 transcripts, and Muc19 knock-out mice mimic the sld phenotype in neonates. Muc19 transcription is unaffected in sld mice, whereas mRNA stability is markedly decreased. Decreased mRNA stability is not due to a defect in 3'-end processing nor to sequence differences in Muc19 transcripts. Comparative sequencing of the Muc19/Smgc gene identified four candidate intronic mutations within the Muc19 coding region. Minigene splicing assays revealed a novel splicing event in which insertion of two additional repeats within a CA repeat region of intron 53 of the sld genome enhances retention of intron 54, decreasing the levels of correctly spliced transcripts. Moreover, pateamine A, an inhibitor of nonsense-mediated mRNA decay, inhibits degradation of aberrant Muc19 transcripts. The mutation in intron 53 thus enhances aberrant splicing leading to degradation of aberrant transcripts and decreased Muc19 message stability, consistent with the sld phenotype. We propose a working model of the unique splicing event enhanced by the mutation, as well as putative explanations for the gradual but limited increase in Muc19 glycoprotein expression and its restricted localization to subpopulations of mucous cells in sld mice during postnatal gland development.

Tissue distibution of murine Muc19/smgc gene products.

The recently identified gene Muc19/Smgc encodes two diverse splice variants, Smgc (submandibular gland protein C) and Muc19 (mucin 19). Muc19 is a member of the large gel-forming mucin family and is an exocrine product of sublingual mucous salivary glands in mice. SMGC is a transiently expressed secretion product of developing rodent submandibular and sublingual glands. Little is known about the expression of Muc19/Smgc gene products in other murine salivary and non-salivary tissues containing the mucous cell phenotype. Muc19 expression was therefore initially assessed by RT-PCR and immunohistochemistry. As a complementary approach, we developed a knockin mouse model, Muc19-EGFP, in which mice express a fusion protein containing the first 69 residues of Muc19 followed by enhanced green fluorescent protein (EGFP) as a marker of Muc19 expression. Results from both approaches are consistent, with preferential Muc19 expression in salivary major and minor mucous glands as well as submucosal glands of the tracheolarynx and bulbourethral glands. Evidence also indicates that individual mucous cells of minor salivary and bulbourethral glands produce another gel-forming mucin in addition to Muc19. We further find tissue expression of full-length Smgc transcripts, which encode for SMGC, and are restricted to neonatal tracheolarynx and all salivary tissues.

Expression of Muc19/Smgc gene products during murine sublingual gland development: cytodifferentiation and maturation of salivary mucous cells.

Muc19/Smgc expresses two splice variants, Smgc (submandibular gland protein C) and Muc19 (mucin 19), the latter a major exocrine product of differentiated murine sublingual mucous cells. Transcripts for Smgc were detected recently in neonatal sublingual glands, suggesting that SMGC proteins are expressed during initial salivary mucous cell cytodifferentiation. We therefore compared developmental expression of transcripts and translation products of Smgc and Muc19 in sublingual glands. We find abundant expression of SMGC within the initial terminal bulbs, with a subsequent decrease as Muc19 expression increases. During postnatal gland expansion, SMGC is found in presumptive newly formed acinar cells and then persists in putative acinar stem cells. Mucin levels increase 7-fold during the first 3 weeks of life, with little change in transcript levels, whereas between postnatal days 21 and 28, there is a 3-fold increase in Muc19 mRNA and heteronuclear RNA. Our collective results demonstrate the direct transition from SMGC to Muc19 expression during early mucous cell cytodifferentiation and further indicate developmentally regulated changes in Muc19/Smgc transcription, alternative splicing, and translation. These changes in Muc19/Smgc gene expression delineate multiple stages of salivary mucous cell cytodifferentiation and subsequent maturation during embryonic gland development through the first 4 weeks of postnatal life.

Mutant LYS2 mRNAs retained and degraded in the nucleus of Saccharomyces cerevisiae.

We previously demonstrated that mRNAs retained in the nucleus of Saccharomyces cerevisiae are subjected to a degradation system-designated DRN (degradation of mRNA in the nucleus), that is diminished in cbc1-Delta or cbc2-Delta mutants lacking components of the cap-binding complex and in rrp6-Delta mutants lacking Rrp6p, a 3' to 5' nuclear exonuclease. Two mutants, lys2-187 and lys2-121, were uncovered by screening numerous lys2 mutants for suppression by cbc1-Delta and rrp6-Delta. Both mutants were identical and contained the two base changes, one of which formed a TGA nonsense codon. LYS2 mRNAs from the lys2-187 and related mutants were rapidly degraded, and the degradation was suppressed by cbc1-Delta and rrp6-Delta. The U1A-GFP imaging procedure was used to show that the lys2-187 mRNA was partially retained in the nucleus, explaining the susceptibility to DRN. The creation of several derivatives of lys2-187 by site-directed mutagenesis revealed that the in-frame TGA by itself was not responsible for the increased susceptibility to DRN. Thus, mRNAs susceptible to DRN can be formed by a 2-bp change. Furthermore, this "retention signal" causing susceptibility to DRN is lost by altering one of the base pairs, establishing that mRNAs susceptible and unsusceptible to DRN can be attributed to a single nucleotide in the proper context.