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1.
Mol Cell ; 81(11): 2388-2402.e8, 2021 06 03.
Article in English | MEDLINE | ID: mdl-33852894

ABSTRACT

Small RNA pathways defend the germlines of animals against selfish genetic elements, yet pathway activities need to be contained to prevent silencing of self genes. Here, we reveal a proteolytic mechanism that controls endogenous small interfering (22G) RNA activity in the Caenorhabditis elegans germline to protect genome integrity and maintain fertility. We find that DPF-3, a P-granule-localized N-terminal dipeptidase orthologous to mammalian dipeptidyl peptidase (DPP) 8/9, processes the unusually proline-rich N termini of WAGO-1 and WAGO-3 Argonaute (Ago) proteins. Without DPF-3 activity, these WAGO proteins lose their proper complement of 22G RNAs. Desilencing of repeat-containing and transposon-derived transcripts, DNA damage, and acute sterility ensue. These phenotypes are recapitulated when WAGO-1 and WAGO-3 are rendered resistant to DPF-3-mediated processing, identifying them as critical substrates of DPF-3. We conclude that N-terminal processing of Ago proteins regulates their activity and promotes silencing of selfish genetic elements by ensuring Ago association with appropriate small RNAs.


Subject(s)
Argonaute Proteins/genetics , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans/genetics , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/genetics , Protein Processing, Post-Translational , RNA, Helminth/genetics , Animals , Argonaute Proteins/metabolism , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/metabolism , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/metabolism , Fertility/genetics , Proteolysis , RNA, Helminth/antagonists & inhibitors , RNA, Helminth/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Substrate Specificity
2.
EMBO J ; 31(19): 3935-48, 2012 Oct 03.
Article in English | MEDLINE | ID: mdl-23032188

ABSTRACT

The Nrd1-Nab3-Sen1 (NNS) complex pathway is responsible for transcription termination of cryptic unstable transcripts and sn/snoRNAs. The NNS complex recognizes short motifs on the nascent RNA, but the presence of these sequences alone is not sufficient to define a functional terminator. We generated a homogeneous set of several hundreds of artificial, NNS-dependent terminators with an in vivo selection approach. Analysis of these terminators revealed novel and extended sequence determinants for transcription termination and NNS complex binding as well as supermotifs that are critical for termination. Biochemical and structural data revealed that affinity and specificity of RNA recognition by Nab3p relies on induced fit recognition implicating an α-helical extension of the RNA recognition motif. Interestingly, the same motifs can be recognized by the NNS or the mRNA termination complex depending on their position relative to the start of transcription, suggesting that they function as general transcriptional insulators to prevent interference between the non-coding and the coding yeast transcriptomes.


Subject(s)
DNA Helicases/metabolism , Gene Expression Regulation, Fungal , Nuclear Proteins/metabolism , RNA Helicases/metabolism , RNA-Binding Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Transcription Termination, Genetic , Amino Acid Motifs/physiology , Amino Acid Sequence , DNA Helicases/chemistry , Molecular Sequence Data , Nuclear Proteins/chemistry , Protein Binding , RNA Helicases/chemistry , RNA-Binding Proteins/chemistry , SELEX Aptamer Technique , Saccharomyces cerevisiae Proteins/chemistry
3.
Mol Cell ; 48(3): 409-21, 2012 Nov 09.
Article in English | MEDLINE | ID: mdl-23000176

ABSTRACT

The exosome is a complex involved in the maturation of rRNA and sn-snoRNA, in the degradation of short-lived noncoding RNAs, and in the quality control of RNAs produced in mutants. It contains two catalytic subunits, Rrp6p and Dis3p, whose specific functions are not fully understood. We analyzed the transcriptome of combinations of Rrp6p and Dis3p catalytic mutants by high-resolution tiling arrays. We show that Dis3p and Rrp6p have both overlapping and specific roles in degrading distinct classes of substrates. We found that transcripts derived from more than half of intron-containing genes are degraded before splicing. Surprisingly, we also show that the exosome degrades large amounts of tRNA precursors despite the absence of processing defects. These results underscore the notion that large amounts of RNAs produced in wild-type cells are discarded before entering functional pathways and suggest that kinetic competition with degradation proofreads the efficiency and accuracy of processing.


Subject(s)
Exosome Multienzyme Ribonuclease Complex/metabolism , RNA Precursors/metabolism , RNA Processing, Post-Transcriptional , Saccharomyces cerevisiae Proteins/metabolism , Blotting, Northern , Exosome Multienzyme Ribonuclease Complex/genetics , Gene Expression Profiling , Introns/genetics , Mutation , Oligonucleotide Array Sequence Analysis , RNA Precursors/genetics , RNA, Fungal/genetics , RNA, Fungal/metabolism , RNA, Ribosomal/genetics , RNA, Ribosomal/metabolism , RNA, Small Nucleolar/genetics , RNA, Small Nucleolar/metabolism , RNA, Transfer/genetics , RNA, Transfer/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Substrate Specificity
4.
EMBO J ; 31(10): 2427-37, 2012 May 16.
Article in English | MEDLINE | ID: mdl-22505027

ABSTRACT

RNA Pol II transcription termination can occur by at least two alternative pathways. Cleavage and polyadenylation by the CPF/CF complex precedes mRNA transcription termination, while the Nrd1 complex is involved in transcription termination of non-coding RNAs such as sno/snRNAs or cryptic unstable transcripts. Here we show that transcription of RPL9B, one of the two genes coding for the ribosomal protein Rpl9p, terminates by either of these two pathways. The balance between these two pathways is modulated in response to the RPL9 gene copy number, resulting in the autoregulation of RPL9B gene expression. This autoregulation mechanism requires a conserved potential stem-loop structure very close to the polyadenylation sites. We propose a model in which Rpl9p, when in excess, binds this conserved 3'-UTR structure, negatively interfering with cleavage and polyadenylation to the benefit of the Nrd1-dependent termination pathway, which, being coupled to degradation by the nuclear exosome, results in downregulation of RPL9B gene expression.


Subject(s)
Gene Expression Regulation, Fungal , Ribosomal Proteins/biosynthesis , Saccharomyces cerevisiae/physiology , Transcription, Genetic , Base Sequence , Models, Biological , Molecular Sequence Data , Nucleic Acid Conformation , Saccharomyces cerevisiae/genetics
5.
Cell ; 135(2): 308-21, 2008 Oct 17.
Article in English | MEDLINE | ID: mdl-18957205

ABSTRACT

During transcription, proteins assemble sequentially with nascent RNA to generate a messenger ribonucleoprotein particle (mRNP). The THO complex and its associated Sub2p helicase are functionally implicated in both transcription and mRNP biogenesis but their precise function remains elusive. We show here that THO/Sub2p mutation leads to the accumulation of a stalled intermediate in mRNP biogenesis that contains nuclear pore components and polyadenylation factors in association with chromatin. Microarray analyses of genomic loci that are aberrantly docked to the nuclear pore in mutants allowed the identification of approximately 400 novel validated target genes that require THO /Sub2p for efficient expression. Our data strongly suggests that the THO complex/Sub2p function is required to coordinate events leading to the acquisition of export competence at a step that follows commitment to 3'-processing.


Subject(s)
Adenosine Triphosphatases/metabolism , Nuclear Pore/metabolism , RNA 3' End Processing , RNA Transport , Saccharomyces cerevisiae/metabolism , Active Transport, Cell Nucleus , Adenosine Triphosphatases/genetics , Chromatin/metabolism , Heat-Shock Proteins/genetics , Mutation , Nuclear Proteins/metabolism , Nucleocytoplasmic Transport Proteins/metabolism , Nucleosomes/metabolism , RNA Polymerase II/metabolism , RNA, Fungal/metabolism , RNA-Binding Proteins/metabolism , Ribonucleoproteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Transcription, Genetic
6.
Nat Struct Mol Biol ; 15(8): 786-94, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18660821

ABSTRACT

Cryptic unstable transcripts (CUTs) are short, 300-600-nucleotide (nt) RNA polymerase II transcripts that are rapidly degraded by the nuclear RNA exosome in yeast. CUTs are widespread and probably represent the largest share of hidden transcription in the yeast genome. Similarly to small nucleolar and small nuclear RNAs, transcription of CUT-encoding genes is terminated by the Nrd1 complex pathway. We show here that this termination mode and ensuing CUTs degradation crucially depend on the position of RNA polymerase II relative to the transcription start site. Notably, position sensing correlates with the phosphorylation status of the polymerase C-terminal domain (CTD). The Nrd1 complex is recruited to chromatin via interactions with both the nascent RNA and the CTD, but a permissive phosphorylation status of the latter is absolutely required for efficient transcription termination. We discuss the mechanism underlying the regulation of coexisting cryptic and mRNA-productive transcription.


Subject(s)
Fungal Proteins/chemistry , Mutation , RNA Polymerase II/chemistry , Ribonucleoproteins/chemistry , Transcription, Genetic , Binding Sites , Chromatin/metabolism , Chromosome Mapping , Fungal Proteins/metabolism , Genome, Fungal , Humans , Kluyveromyces/metabolism , Phosphorylation , Protein Structure, Tertiary , RNA/chemistry , RNA, Small Nuclear/chemistry , Ribonucleoproteins/metabolism , Saccharomyces cerevisiae
7.
Biol Cell ; 100(6): 327-42, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18479253

ABSTRACT

In eukaryotes, copying the genetic information from a DNA template into RNA is not sufficient itself to confer functional competence to the DNA-encoded message. mRNAs have to be processed by enzymes and packaged with proteins within nuclei to generate mRNP (messenger ribonucleoprotein) particles, before these can be exported to the cytoplasm. Processing and packaging factors are believed to interact with the nascent mRNA co-transcriptionally, which protects the highly reactive RNA molecule from a presumably aggressive nuclear environment while providing early commitment to its functional fate. In this review, we will describe the factors that are believed to provide the appropriate 'dress code' to the mRNA and the mechanisms underlying the proofreading events that guarantee its quality, focusing on yeast as a model system.


Subject(s)
Cytoplasm/metabolism , RNA Processing, Post-Transcriptional , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Ribonucleoproteins/metabolism , Yeasts/metabolism , Cell Nucleus/genetics , Cell Nucleus/metabolism , Cytoplasm/genetics , Fungal Proteins/genetics , Fungal Proteins/metabolism , Genes, cdc , RNA Transport , RNA, Messenger/genetics , RNA-Binding Proteins/genetics , Ribonucleoproteins/genetics , Transcription, Genetic , Yeasts/genetics
8.
EMBO J ; 26(9): 2317-26, 2007 May 02.
Article in English | MEDLINE | ID: mdl-17410208

ABSTRACT

The nuclear exosome is involved in numerous RNA metabolic processes. Exosome degradation of rRNA, snoRNA, snRNA and tRNA in Saccharomyces cerevisiae is activated by TRAMP complexes, containing either the Trf4p or Trf5p poly(A) polymerase. These enzymes are presumed to facilitate exosome access by appending oligo(A)-tails onto structured substrates. Another role of the nuclear exosome is that of mRNA surveillance. In strains harboring a mutated THO/Sub2p system, involved in messenger ribonucleoprotein particle biogenesis and nuclear export, the exosome-associated 3' --> 5' exonuclease Rrp6p is required for both retention and degradation of nuclear restricted mRNAs. We show here that Trf4p, in the context of TRAMP, is an mRNA surveillance factor. However, unlike Rrp6p, Trf4p only partakes in RNA degradation and not in transcript retention. Surprisingly, a polyadenylation-defective Trf4p protein is fully active, suggesting polyadenylation-independent mRNA degradation. Transcription pulse-chase experiments show that HSP104 molecules undergoing quality control in THO/sub2 mutant strains fall into two distinct populations: One that is quickly degraded after transcription induction and another that escapes rapid decay and accumulates in foci associated with the HSP104 transcription site.


Subject(s)
Adenosine Triphosphatases/metabolism , Cell Nucleus/metabolism , DNA-Binding Proteins/metabolism , RNA, Messenger/metabolism , Ribonucleoproteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Transcription Factors/metabolism , Adenosine Triphosphatases/genetics , DNA-Binding Proteins/genetics , DNA-Directed DNA Polymerase/metabolism , Exoribonucleases/metabolism , Exosome Multienzyme Ribonuclease Complex , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Multiprotein Complexes , Mutation , Nuclear Proteins , Polyadenylation , RNA Stability/physiology , Ribonucleoproteins/genetics , Saccharomyces cerevisiae Proteins/genetics , Transcription Factors/genetics , Transcription, Genetic
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