Molecular basis for the distinct cellular functions of the Lsm1–7 and Lsm2–8 complexes

Eric, Montemayor,; Virta, Johanna M; Hayes, Samuel M; Nomura, Yoshinobu; Brow, David A.; Butcher, Samuel E.

doi:10.1261/rna.075879.120

Cited by 25 publications

(33 citation statements)

References 74 publications

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“…S2). As this work was in review, a crystal structure of S. pombe Lsm1-7 with U5A1 RNA was reported, demonstrating binding to the distal face of the ring, similar to the Lsm2-8 complex (Montemayor et al 2020). Determining the binding modes of different RNAs with the Pat1/Lsm1-7 complex remains a challenge for future structural studies.…”

Section: Discussionmentioning

confidence: 91%

Pdc2/Pat1 increases the range of decay factors and RNA bound by the Lsm1–7 complex

Lobel

Gross

2020

RNA

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Pat1 promotes the activation and assembly of multiple proteins during mRNA decay. After deadenylation, the Pat1/Lsm1-7 complex binds to transcripts containing oligo(A) tails, which can be modified by the addition of several terminal uridine residues. Pat1 enhances Lsm1-7 binding to the 3' end, but it is unknown how this interaction is influenced by nucleotide composition. Here we examine Pat1/Lsm1-7 binding to a series of oligoribonucleotides containing different A/U contents using recombinant purified proteins from fission yeast. We observe a positive correlation between fractional uridine content and Lsm1-7 binding affinity. Addition of Pat1 broadens RNA specificity of Lsm1-7 by enhancing binding to A-rich RNAs and increases cooperativity on all oligonucleotides tested. Consistent with increased cooperativity, Pat1 promotes multimerization of the Lsm1-7 complex, which is potentiated by RNA binding. Furthermore, the inherent ability of Pat1 to multimerize drives liquid-liquid phase separation with multivalent decapping enzyme complexes of Dcp1/Dcp2. Our results uncover how Pat1 regulates RNA binding and higher order assembly by mRNA decay factors.

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Section: Discussionmentioning

confidence: 91%

Pdc2/Pat1 increases the range of decay factors and RNA bound by the Lsm1–7 complex

Lobel

Gross

2020

RNA

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“…In S. pombe and mammalian cells, uridylation is proposed to favor decapping of deadenylated mRNA by promoting the binding of the LSm1-7 complex 5 , 8 , 53 , which recruits the decapping complex through a connection with Pat1 54 – 57 . The LSm1-7 complex preferentially binds short oligo(A) tails of <10 As 23 .…”

Section: Discussionmentioning

confidence: 99%

The TUTase URT1 connects decapping activators and prevents the accumulation of excessively deadenylated mRNAs to avoid siRNA biogenesis

et al. 2021

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Uridylation is a widespread modification destabilizing eukaryotic mRNAs. Yet, molecular mechanisms underlying TUTase-mediated mRNA degradation remain mostly unresolved. Here, we report that the Arabidopsis TUTase URT1 participates in a molecular network connecting several translational repressors/decapping activators. URT1 directly interacts with DECAPPING 5 (DCP5), the Arabidopsis ortholog of human LSM14 and yeast Scd6, and this interaction connects URT1 to additional decay factors like DDX6/Dhh1-like RNA helicases. Nanopore direct RNA sequencing reveals a global role of URT1 in shaping poly(A) tail length, notably by preventing the accumulation of excessively deadenylated mRNAs. Based on in vitro and in planta data, we propose a model that explains how URT1 could reduce the accumulation of oligo(A)-tailed mRNAs both by favoring their degradation and because 3’ terminal uridines intrinsically hinder deadenylation. Importantly, preventing the accumulation of excessively deadenylated mRNAs avoids the biogenesis of illegitimate siRNAs that silence endogenous mRNAs and perturb Arabidopsis growth and development.

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“…Sample heterogeneity, oligomerization, and the identification of a functionally relevant conformer represent long-standing challenges in the analysis of biochemical data. Dimerization and higher order oligomers of RNA binding proteins can function as essential features for splicing regulation, posttranscriptional processing, and RNA biogenesis, or they can represent aberrant pathways prone to aggregation that can dominate the pathology of a disease (Lagier-Tourenne et al, 2010;Couthouis et al, 2011;Prusty et al, 2017;Montalbano et al, 2020;Montemayor et al, 2020). In bacterial RNase P, the issue of a functional dimeric holoenzyme has been extensively discussed (Fang et al, 2001;Barrera et al, 2002;Barrera and Pan 2004;Buck et al, 2005a;Niland et al, 2017), though it has been structurally validated that the RNA-protein holoenzyme complex in bacteria and eukaryotes function as single, monomeric assemblies to perform ptRNA recognition and catalysis.…”

Section: Discussionmentioning

confidence: 99%

Dissecting Monomer-Dimer Equilibrium of an RNase P Protein Provides Insight Into the Synergistic Flexibility of 5’ Leader Pre-tRNA Recognition

Zeng

Abzhanova

Brown

et al. 2021

Front. Mol. Biosci.

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Ribonuclease P (RNase P) is a universal RNA-protein endonuclease that catalyzes 5’ precursor-tRNA (ptRNA) processing. The RNase P RNA plays the catalytic role in ptRNA processing; however, the RNase P protein is required for catalysis in vivo and interacts with the 5’ leader sequence. A single P RNA and a P protein form the functional RNase P holoenzyme yet dimeric forms of bacterial RNase P can interact with non-tRNA substrates and influence bacterial cell growth. Oligomeric forms of the P protein can also occur in vitro and occlude the 5’ leader ptRNA binding interface, presenting a challenge in accurately defining the substrate recognition properties. To overcome this, concentration and temperature dependent NMR studies were performed on a thermostable RNase P protein from Thermatoga maritima. NMR relaxation (R1, R2), heteronuclear NOE, and diffusion ordered spectroscopy (DOSY) experiments were analyzed, identifying a monomeric species through the determination of the diffusion coefficients (D) and rotational correlation times (τc). Experimental diffusion coefficients and τc values for the predominant monomer (2.17 ± 0.36 * 10−10 m2/s, τc = 5.3 ns) or dimer (1.87 ± 0.40* 10−10 m2/s, τc = 9.7 ns) protein assemblies at 45°C correlate well with calculated diffusion coefficients derived from the crystallographic P protein structure (PDB 1NZ0). The identification of a monomeric P protein conformer from relaxation data and chemical shift information enabled us to gain novel insight into the structure of the P protein, highlighting a lack of structural convergence of the N-terminus (residues 1–14) in solution. We propose that the N-terminus of the bacterial P protein is partially disordered and adopts a stable conformation in the presence of RNA. In addition, we have determined the location of the 5’ leader RNA in solution and measured the affinity of the 5’ leader RNA–P protein interaction. We show that the monomer P protein interacts with RNA at the 5’ leader binding cleft that was previously identified using X-ray crystallography. Data support a model where N-terminal protein flexibility is stabilized by holoenzyme formation and helps to accommodate the 5’ leader region of ptRNA. Taken together, local structural changes of the P protein and the 5’ leader RNA provide a means to obtain optimal substrate alignment and activation of the RNase P holoenzyme.

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Molecular basis for the distinct cellular functions of the Lsm1–7 and Lsm2–8 complexes

Cited by 25 publications

References 74 publications

Pdc2/Pat1 increases the range of decay factors and RNA bound by the Lsm1–7 complex

Pdc2/Pat1 increases the range of decay factors and RNA bound by the Lsm1–7 complex

The TUTase URT1 connects decapping activators and prevents the accumulation of excessively deadenylated mRNAs to avoid siRNA biogenesis

Dissecting Monomer-Dimer Equilibrium of an RNase P Protein Provides Insight Into the Synergistic Flexibility of 5’ Leader Pre-tRNA Recognition

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