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Role of Has1 RNA helicase in ribosome biogenesis in yeast S. cerevisiae

ABSTRACT: The RNA helicase Has1 is involved in the biogenesis of both small and large ribosomal subunit. How it performs these separate roles is not fully understood. We analysed a detailed composition of RNA-protein complexes purified via Has1.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Martin Kos

LAB HEAD: Martin Koš

PROVIDER: PXD013263 | Pride | 2019-09-09

REPOSITORIES: Pride

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			Action	DRS
	Has1-AAA100NaCl_12-232-13.raw	Raw
	Has1-AAA100NaCl_12-232-14.raw	Raw
	Has1-AAA100NaCl_12-232-15.raw	Raw
	Has1-AAA500NaCl_12-232-17.raw	Raw
	Has1-AAA500NaCl_12-232-18.raw	Raw

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Publications

At least two molecules of the RNA helicase Has1 are simultaneously present in pre-ribosomes during ribosome biogenesis.

Gnanasundram Sivakumar Vadivel SV Kos-Braun Isabelle C IC Koš Martin M

Nucleic acids research 20191101 20

The RNA helicase Has1 is involved in the biogenesis of both small and large ribosomal subunits. How it performs these separate roles is not fully understood. Here we provide evidence that at least two molecules of Has1 are temporarily present at the same time in 90S pre-ribosomes. We identified multiple Has1 binding sites in the 18S, 5.8S and 25S rRNAs. We show that while the Has1 catalytic activity is not required for binding to 5.8S/25S region in pre-rRNA, it is essential for binding to 18S si ...[more]

PMID: 31511893

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Project description:Emerging evidence suggests that Ago/Piwi proteins function in the nucleus as well as the cytoplasm to control gene expression, but the mechanisms they employ in the nucleus remain poorly defined. The Tetrahymena thermophila Ago/Piwi protein Twi12 is essential for growth and functions in the nucleus. We show that Twi12 interacts with the exonuclease Xrn2. Twi12 functions to stabilize and localize Xrn2 in vivo, as well as activate its exonuclease activity in vitro. When Twi12 or Xrn2 are depleted, pre-rRNA processing intermediates accumulate and mature rRNA levels decline. RNA polymerase I and II (RNAP I and II) transcripts also accumulate. Twi12 function depends on small RNA (sRNA) binding, which is required for its nuclear import. Twi12-bound sRNAs are Dicer-independent 3' tRNA fragments that we propose may not be involved in sequence-specific base pairing to targets. Our findings suggest a role for tRNA fragments and an Ago/Piwi protein in global control of gene expression through interaction with a conserved exonuclease. One library is analyzed here: small RNAs associated with ZZF-tagged Twi12. Twi12 is the full-length version, which is extended by 17 amino acids at the N-terminus compared to the previously-reported Twi12. Specifically, the 16-22 nt reads were analyzed here.

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Project description:During ribosome biogenesis a plethora of assembly factors and essential enzymes drive the unidirectional maturation of nascent pre-ribosomal subunits. The DEAD-box RNA helicase Dbp10 is suggested to restructure pre-ribosomal rRNA of the evolving peptidyl-transferase center (PTC) on nucleolar ribosomal 60S assembly intermediates. Here, we show that point mutations within conserved catalytic helicase-core motifs of Dbp10 yield a dominant-lethal growth phenotype. Such dbp10 mutants, which stably associate with pre-60S intermediates, impair pre-60S biogenesis at a nucleolar stage prior to the release of assembly factor Rrp14 and stable integration of late nucleolar factors such as Noc3. Furthermore, the binding of the GTPase Nug1 to particles isolated directly via mutant Dbp10 bait proteins is specifically inhibited. The N-terminal domain of Nug1 interacts with Dbp10 and the methyltransferase Spb1, whose pre-60S incorporation is also reduced in absence of functional Dbp10. Our data suggest that Dbp10’s helicase activity generates a crucial framework for assembly factor docking thereby permitting the progression of pre-60S maturation

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Dbp10p, a putative RNA helicase from Saccharomyces cerevisiae, is required for ribosome biogenesis.

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