Inactivation of fission yeast Erh1 de-represses pho1 expression: evidence that Erh1 is a negative regulator of prt lncRNA termination.

Fission yeast Erh1 exists in a complex with RNA-binding protein Mmi1. Deletion of erh1 up-regulates the phosphate homeostasis gene pho1, which is normally repressed by transcription in cis of a 5' flanking prt lncRNA. Here we present evidence that de-repression of pho1 by erh1Δ is achieved through precocious 3'-processing/termination of prt lncRNA synthesis, to wit: (i) erh1Δ does not affect the activity of the prt or pho1 promoters per se; (ii) de-repression by erh1Δ depends on CPF (cleavage and polyadenylation factor) subunits Ctf1, Dis2, Ssu72, Swd22, and Ppn1 and on termination factor Rhn1; (iii) de-repression requires synthesis by the Asp1 IPP kinase of inositol 1-pyrophosphates (1-IPPs); (iv) de-repression is effaced by mutating Thr4 of the RNA polymerase II CTD to alanine; and (v) erh1Δ exerts an additive effect on pho1 de-repression in combination with mutating CTD Ser7 to alanine and with deletion of the IPP pyrophosphatase Aps1. These findings point to Erh1 as an antagonist of lncRNA termination in the prt-pho1 axis. In contrast, in mmi1Δ cells there is a reduction in pho1 mRNA and increase in the formation of a prt-pho1 read-through transcript, consistent with Mmi1 being an agonist of prt termination. We envision that Erh1 acts as a brake on Mmi1's ability to promote CPF-dependent termination during prt lncRNA synthesis. Consistent with this idea, erh1Δ de-repression of pho1 was eliminated by mutating the Mmi1-binding sites in the prt lncRNA.

Relative contributions of the structural and catalytic roles of Rrp6 in exosomal degradation of individual mRNAs.

The RNA exosome is a conserved complex for RNA degradation with two ribonucleolytic subunits, Dis3 and Rrp6. Rrp6 is a 3'-5' exonuclease, but it also has a structural role in helping target RNAs to the Dis3 activity. The relative importance of the exonuclease activity and the targeting activity probably differs between different RNA substrates, but this is poorly understood. To understand the relative contributions of the exonuclease and the targeting activities to the degradation of individual RNA substrates in Schizosaccharomyces pombe, we compared RNA levels in an rrp6 null mutant to those in an rrp6 point mutant specifically defective in exonuclease activity. A wide range of effects was found, with some RNAs dependent mainly on the structural role of Rrp6 ("protein-dependent" targets), other RNAs dependent mainly on the catalytic role ("activity-dependent" targets), and some RNAs dependent on both. Some protein-dependent RNAs contained motifs targeted via the RNA-binding protein Mmi1, while others contained a motif possibly involved in response to iron. In these and other cases Rrp6 may act as a structural adapter to target specific RNAs to the exosome by interacting with sequence-specific RNA-binding proteins.

Structural insights into the specific recognition of DSR by the YTH domain containing protein Mmi1.

Meiosis is one of the most dramatic differentiation programs accompanied by a striking change in gene expression profiles in fission yeast Schizosaccharomyces pombe. Whereas a number of meiosis-specific transcripts are expressed untimely in mitotic cells, and the entry of meiosis will be blocked as the accumulation of meiosis-specific mRNAs in the mitotic cells. A YTH domain containing protein Mmi1 was identified as a pivotal effector in a post-transcriptional event termed selective elimination of meiosis-specific mRNAs. Mmi1 can recognize and bind a class of meiosis-specific transcripts expressed inappropriately in mitotic cells, which all contain a conservative region called DSR, as a mark to remove them in cooperation with nuclear exosomes. Here we report the 1.6 Å resolution crystal structure of the Mmi1-YTH domain in complex with a high consensus hexanucleotide motif, which is multiple copied in the DSR region. Our structure observations, supported by site-directed mutations of key residues illustrate the mechanism for specific recognition of DSR-RNA by Mmi1. Moreover, different from other YTH domain family proteins, Mmi1-YTH domain has a distinctive RNA-binding properties although it has a similar fold as other ones.

Mmi1, the Yeast Ortholog of Mammalian Translationally Controlled Tumor Protein (TCTP), Negatively Affects Rapamycin-Induced Autophagy in Post-Diauxic Growth Phase.

Translationally controlled tumor protein (TCTP) is a multifunctional and highly conserved protein from yeast to humans. Recently, its role in non-selective autophagy has been reported with controversial results in mammalian and human cells. Herein we examine the effect of Mmi1, the yeast ortholog of TCTP, on non-selective autophagy in budding yeast Saccharomyces cerevisiae, a well-established model system to monitor autophagy. We induced autophagy by nitrogen starvation or rapamycin addition and measured autophagy by using the Pho8Δ60 and GFP-Atg8 processing assays in WT, mmi1Δ, and in autophagy-deficient strains atg8Δ or atg1Δ. Our results demonstrate that Mmi1 does not affect basal or nitrogen starvation-induced autophagy. However, an increased rapamycin-induced autophagy is detected in mmi1Δ strain when the cells enter the post-diauxic growth phase, and this phenotype can be rescued by inserted wild-type MMI1 gene. Further, the mmi1Δ cells exhibit significantly lower amounts of reactive oxygen species (ROS) in the post-diauxic growth phase compared to WT cells. In summary, our study suggests that Mmi1 negatively affects rapamycin-induced autophagy in the post-diauxic growth phase and supports the role of Mmi1/TCTP as a negative autophagy regulator in eukaryotic cells.

Dense Transposon Integration Reveals Essential Cleavage and Polyadenylation Factors Promote Heterochromatin Formation.

Heterochromatin functions as a scaffold for factors responsible for gene silencing and chromosome segregation. Heterochromatin can be assembled by multiple pathways, including RNAi and RNA surveillance. We identified factors that form heterochromatin using dense profiles of transposable element integration in Schizosaccharomyces pombe. The candidates include a large number of essential proteins such as four canonical mRNA cleavage and polyadenylation factors. We find that Iss1, a subunit of the poly(A) polymerase module, plays a role in forming heterochromatin in centromere repeats that is independent of RNAi. Genome-wide maps reveal that Iss1 accumulates at genes regulated by RNA surveillance. Iss1 interacts with RNA surveillance factors Mmi1 and Rrp6, and importantly, Iss1 contributes to RNA elimination that forms heterochromatin at meiosis genes. Our profile of transposable element integration supports the model that a network of mRNA cleavage and polyadenylation factors coordinates RNA surveillance, including the mechanism that forms heterochromatin at meiotic genes.

CPF Recruitment to Non-canonical Transcription Termination Sites Triggers Heterochromatin Assembly and Gene Silencing.

In eukaryotic genomes, heterochromatin is targeted by RNAi machinery and/or by pathways requiring RNA elimination and transcription termination factors. However, a direct connection between termination machinery and RNA polymerase II (RNAPII) transcriptional activity at heterochromatic loci has remained elusive. Here, we show that, in fission yeast, the conserved cleavage and polyadenylation factor (CPF) is a key component involved in RNAi-independent assembly of constitutive and facultative heterochromatin domains and that CPF is broadly required to silence genes regulated by Clr4SUV39H. Remarkably, CPF is recruited to non-canonical termination sites within the body of genes by the YTH family RNA-binding protein Mmi1 and is required for RNAPII transcription termination and facultative heterochromatin assembly. CPF loading by Mmi1 also promotes the selective termination of long non-coding RNAs that regulate gene expression in cis. These analyses delineate a mechanism in which CPF loaded onto non-canonical termination sites specifies targets of heterochromatin assembly and gene silencing.

mmi1 and rep2 mRNAs are novel RNA targets of the Mei2 RNA-binding protein during early meiosis in Schizosaccharomyces pombe.

The RNA-binding protein Mei2 is crucial for meiosis in Schizosaccharomyces pombe. In mei2 mutants, pre-meiotic S-phase is blocked, along with meiosis. Mei2 binds a long non-coding RNA (lncRNA) called meiRNA, which is a 'sponge RNA' for the meiotic inhibitor protein Mmi1. The interaction between Mei2, meiRNA and Mmi1 protein is essential for meiosis. But mei2 mutants have stronger and different phenotypes than meiRNA mutants, since mei2Δ arrests before pre-meiotic S, while the meiRNA mutant arrests after pre-meiotic S but before meiosis. This suggests Mei2 may bind additional RNAs. To identify novel RNA targets of Mei2, which might explain how Mei2 regulates pre-meiotic S, we used RNA immunoprecipitation and cross-linking immunoprecipitation. In addition to meiRNA, we found the mRNAs for mmi1 (which encodes Mmi1) and for the S-phase transcription factor rep2 There were also three other RNAs of uncertain relevance. We suggest that at meiotic initiation, Mei2 may sequester rep2 mRNA to help allow pre-meiotic S, and then may bind both meiRNA and mmi1 mRNA to inactivate Mmi1 at two levels, the protein level (as previously known), and also the mRNA level, allowing meiosis. We call Mei2-meiRNA a 'double sponge' (i.e. binding both an mRNA and its encoded protein).

Ubiquitination-dependent control of sexual differentiation in fission yeast.

In fission yeast, meiosis-specific transcripts are selectively eliminated during vegetative growth by the combined action of the YTH-family RNA-binding protein Mmi1 and the nuclear exosome. Upon nutritional starvation, the master regulator of meiosis Mei2 inactivates Mmi1, thereby allowing expression of the meiotic program. Here, we show that the E3 ubiquitin ligase subunit Not4/Mot2 of the evolutionarily conserved Ccr4-Not complex, which associates with Mmi1, promotes suppression of meiotic transcripts expression in mitotic cells. Our analyses suggest that Mot2 directs ubiquitination of Mei2 to preserve the activity of Mmi1 during vegetative growth. Importantly, Mot2 is not involved in the constitutive pathway of Mei2 turnover, but rather plays a regulatory role to limit its accumulation or inhibit its function. We propose that Mmi1 recruits the Ccr4-Not complex to counteract its own inhibitor Mei2, thereby locking the system in a stable state that ensures the repression of the meiotic program by Mmi1.

Regulation of mRNA Levels by Decay-Promoting Introns that Recruit the Exosome Specificity Factor Mmi1.

In eukaryotic cells, inefficient splicing is surprisingly common and leads to the degradation of transcripts with retained introns. How pre-mRNAs are committed to nuclear decay is unknown. Here, we uncover a mechanism by which specific intron-containing transcripts are targeted for nuclear degradation in fission yeast. Sequence elements within these "decay-promoting" introns co-transcriptionally recruit the exosome specificity factor Mmi1, which induces degradation of the unspliced precursor and leads to a reduction in the levels of the spliced mRNA. This mechanism negatively regulates levels of the RNA helicase DDX5/Dbp2 to promote cell survival in response to stress. In contrast, fast removal of decay-promoting introns by co-transcriptional splicing precludes Mmi1 recruitment and relieves negative expression regulation. We propose that decay-promoting introns facilitate the regulation of gene expression. Based on the identification of multiple additional Mmi1 targets, including mRNAs, long non-coding RNAs, and sn/snoRNAs, we suggest a general role in RNA regulation for Mmi1 through transcript degradation.

Hexanucleotide motifs mediate recruitment of the RNA elimination machinery to silent meiotic genes.

The selective elimination system blocks the accumulation of meiosis-specific mRNAs during the mitotic cell cycle in fission yeast. These mRNAs harbour a region, the determinant of selective removal (DSR), which is recognized by a YTH-family RNA-binding protein, Mmi1. Mmi1 directs target transcripts to destruction in association with nuclear exosomes. Hence, the interaction between DSR and Mmi1 is crucial to discriminate mitosis from meiosis. Here, we show that Mmi1 interacts with repeats of the hexanucleotide U(U/C)AAAC that are enriched in the DSR. Disruption of this 'DSR core motif' in a target mRNA inhibits its elimination. Tandem repeats of the motif can function as an artificial DSR. Mmi1 binds to it in vitro. Thus, a core motif cluster is responsible for the DSR activity. Furthermore, certain variant hexanucleotide motifs can augment the function of the DSR core motif. Notably, meiRNA, which composes the nuclear Mei2 dot required to suppress Mmi1 activity during meiosis, carries numerous copies of the core/augmenting motifs on its tail and is indeed degraded by the Mmi1/exosome system, indicating its likely role as decoy bait for Mmi1.