Threading the barrel of the RNA exosome

In eukaryotes, the exosome complex degrades RNA backbones and plays key roles in RNA processing and surveillance. It was predicted that RNA substrates are threaded through a central channel. This pathway is conserved between eukaryotic and archaeal complexes, even though nuclease activity was lost from the nine-subunit eukaryotic core (EXO-9) and transferred to associated proteins. The exosome cooperates with nuclear and cytoplasmic cofactors, including RNA helicases Mtr4 and Ski2, respectively. Structures of an RNA-bound exosome and both helicases revealed how substrates are channeled through EXO-9 to the associated nuclease Rrp44. Recent high-throughput analyses provided fresh insights relating exosome structure to its diverse in vivo functions. They also revealed surprisingly high degradation rates for newly synthesized RNAs, particularly RNA polymerase III transcripts

Identification of a regulated pathway for nuclear pre-mRNA turnover

We have identified a nuclear pathway that rapidly degrades unspliced pre-mRNAs in yeast. This involves 3′→5′ degradation by the exosome complex and 5′→3′ degradation by the exonuclease Rat1p. 3′→5′ degradation is normally the major pathway and is regulated in response to carbon source. Inhibition of pre-mRNA degradation resulted in increased levels of pre-mRNAs and spliced mRNAs. When splicing was inhibited by mutation of a splicing factor, inhibition of turnover resulted in 20- to 50-fold accumulation of pre-mRNAs, accompanied by increased mRNA production. Splicing of a reporter construct with a 3′ splice site mutation was also increased on inhibition of turnover, showing competition between degradation and splicing. We propose that nuclear pre-mRNA turnover represents a novel step in the regulation of gene expression

A complex pathway for 3 ' processing of the yeast U3 snoRNA

Mature U3 snoRNA in yeast is generated from the 3′-extended precursors by endonucleolytic cleavage followed by exonucleolytic trimming. These precursors terminate in poly(U) tracts and are normally stabilised by binding of the yeast La homologue, Lhp1p. We report that normal 3′ processing of U3 requires the nuclear Lsm proteins. On depletion of any of the five essential proteins, Lsm2–5p or Lsm8p, the normal 3′-extended precursors to the U3 snoRNA were lost. Truncated fragments of both mature and pre-U3 accumulated in the Lsm-depleted strains, consistent with substantial RNA degradation. Pre-U3 species were co-precipitated with TAP-tagged Lsm3p, but the association with spliced pre-U3 was lost in strains lacking Lhp1p. The association of Lhp1p with pre-U3 was also reduced on depletion of Lsm3p or Lsm5p, indicating that binding of Lhp1p and the Lsm proteins is interdependent. In contrast, a tagged Sm-protein detectably co-precipitated spliced pre-U3 species only in strains lacking Lhp1p. We propose that the Lsm2–8p complex functions as a chaperone in conjunction with Lhp1p to stabilise pre-U3 RNA species during 3′ processing. The Sm complex may function as a back-up to stabilise 3′ ends that are not protected by Lhp1p

The SPARC complex defines RNAPII promoters in Trypanosoma brucei

Kinetoplastids are a highly divergent lineage of eukaryotes with unusual mechanisms for regulating gene expression. We previously surveyed 65 putative chromatin factors in the kinetoplastid Trypanosoma brucei. Our analyses revealed that the predicted histone methyltransferase SET27 and the Chromodomain protein CRD1 are tightly concentrated at RNAPII transcription start regions (TSRs). Here, we report that SET27 and CRD1, together with four previously uncharacterized constituents, form the SET27 promoter-associated regulatory complex (SPARC), which is specifically enriched at TSRs. SET27 loss leads to aberrant RNAPII recruitment to promoter sites, accumulation of polyadenylated transcripts upstream of normal transcription start sites, and conversion of some normally unidirectional promoters to bidirectional promoters. Transcriptome analysis in the absence of SET27 revealed upregulated mRNA expression in the vicinity of SPARC peaks within the main body of chromosomes in addition to derepression of genes encoding variant surface glycoproteins (VSGs) located in subtelomeric regions. These analyses uncover a novel chromatin-associated complex required to establish accurate promoter position and directionality

The DEAH-box helicase Dhr1 dissociates U3 from the pre-rRNA to promote formation of the central pseudoknot

In eukaryotes, the highly conserved U3 small nucleolar RNA (snoRNA) base-pairs to multiple sites in the pre-ribosomal RNA (pre-rRNA) to promote early cleavage and folding events. Binding of the U3 box A region to the pre-rRNA is mutually exclusive with folding of the central pseudoknot (CPK), a universally conserved rRNA structure of the small ribosomal subunit essential for protein synthesis. Here, we report that the DEAH-box helicase Dhr1 (Ecm16) is responsible for displacing U3. An active site mutant of Dhr1 blocked release of U3 from the pre-ribosome, thereby trapping a pre-40S particle. This particle had not yet achieved its mature structure because it contained U3, pre-rRNA, and a number of early-acting ribosome synthesis factors but noticeably lacked ribosomal proteins (r-proteins) that surround the CPK. Dhr1 was cross-linked in vivo to the pre-rRNA and to U3 sequences flanking regions that base-pair to the pre-rRNA including those that form the CPK. Point mutations in the box A region of U3 suppressed a cold-sensitive mutation of Dhr1, strongly indicating that U3 is an in vivo substrate of Dhr1. To support the conclusions derived from in vivo analysis we showed that Dhr1 unwinds U3-18S duplexes in vitro by using a mechanism reminiscent of DEAD box proteins

NANOS2 is a sequence-specific mRNA-binding protein that promotes transcript degradation in spermatogonial stem cells

Summary: Spermatogonial stem cells (SSCs) sustain spermatogenesis and fertility throughout adult male life. The conserved RNA-binding protein NANOS2 is essential for the maintenance of SSCs, but its targets and mechanisms of function are not fully understood. Here, we generated a fully functional epitope-tagged Nanos2 mouse allele and applied the highly stringent cross-linking and analysis of cDNAs to define NANOS2 RNA occupancy in SSC lines. NANOS2 recognizes the AUKAAWU consensus motif, mostly found in the 3′ untranslated region of defined messenger RNAs (mRNAs). We find that NANOS2 is a regulator of key signaling and metabolic pathways whose dosage or activity are known to be critical for SSC maintenance. NANOS2 interacts with components of CCR4-NOT deadenylase complex in SSC lines, and consequently, NANOS2 binding reduces the half-lives of target transcripts. In summary, NANOS2 contributes to SSC maintenance through the regulation of target mRNA stability and key self-renewal pathways