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.

RNA:DNA hybrids from Okazaki fragments contribute to establish the Ku-mediated barrier to replication-fork degradation.

Nonhomologous end-joining (NHEJ) factors act in replication-fork protection, restart, and repair. Here, we identified a mechanism related to RNA:DNA hybrids to establish the NHEJ factor Ku-mediated barrier to nascent strand degradation in fission yeast. RNase H activities promote nascent strand degradation and replication restart, with a prominent role of RNase H2 in processing RNA:DNA hybrids to overcome the Ku barrier to nascent strand degradation. RNase H2 cooperates with the MRN-Ctp1 axis to sustain cell resistance to replication stress in a Ku-dependent manner. Mechanistically, the need of RNaseH2 in nascent strand degradation requires the primase activity that allows establishing the Ku barrier to Exo1, whereas impairing Okazaki fragment maturation reinforces the Ku barrier. Finally, replication stress induces Ku foci in a primase-dependent manner and favors Ku binding to RNA:DNA hybrids. We propose a function for the RNA:DNA hybrid originating from Okazaki fragments in controlling the Ku barrier specifying nuclease requirement to engage fork resection.

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.

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.