The <i>Euplotes</i> telomerase subunit p43 stimulates enzymatic activity and processivity in vitro

Aigner, Stefan; Cech, Thomas R.

doi:10.1261/rna.7400704

Cited by 27 publications

(28 citation statements)

References 52 publications

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“…The phylogenetic analysis of the LAM-RRM (RRM-L) region gives a sharper distinction between these two related sets of proteins and strongly supports this previous classification. We also found that the functionally characterized ciliate p43 (EaLARP7) and p65 (TthLARP7) proteins (Aigner et al 2000(Aigner et al , 2003Aigner and Cech 2004;Witkin and Collins 2004;Stone et al 2007;Teixeira and Gilson 2007) are group 2 factors and cluster with the other protist proteins of this group. As for genuine La proteins, not all group 2 factors possess an RRM2 motif (see PsLARP7 and p65 [TthLARP7]), rendering both structural and evolutionary analyses necessary for classification.…”

Section: Phylogenetic Relationships Among the La-rrm Domains: Co-evolmentioning

confidence: 62%

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A comprehensive analysis of the La-motif protein superfamily

Bousquet‐Antonelli¹,

Deragon²

2009

RNA

166

284

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The extremely well-conserved La motif (LAM), in synergy with the immediately following RNA recognition motif (RRM), allows direct binding of the (genuine) La autoantigen to RNA polymerase III primary transcripts. This motif is not only found on La homologs, but also on La-related proteins (LARPs) of unrelated function. LARPs are widely found amongst eukaryotes and, although poorly characterized, appear to be RNA-binding proteins fulfilling crucial cellular functions. We searched the fully sequenced genomes of 83 eukaryotic species scattered along the tree of life for the presence of LAM-containing proteins. We observed that these proteins are absent from archaea and present in all eukaryotes (except protists from the Plasmodium genus), strongly suggesting that the LAM is an ancestral motif that emerged early after the archaea-eukarya radiation. A complete evolutionary and structural analysis of these proteins resulted in their classification into five families: the genuine La homologs and four LARP families. Unexpectedly, in each family a conserved domain representing either a classical RRM or an RRM-like motif immediately follows the LAM of most proteins. An evolutionary analysis of the LAM-RRM/RRM-L regions shows that these motifs co-evolved and should be used as a single entity to define the functional region of interaction of LARPs with their substrates. We also found two extremely well conserved motifs, named LSA and DM15, shared by LARP6 and LARP1 family members, respectively. We suggest that members of the same family are functional homologs and/or share a common molecular mode of action on different RNA baits.

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Section: Phylogenetic Relationships Among the La-rrm Domains: Co-evolmentioning

confidence: 62%

“…They are key member proteins of the telomerase RNP and are required for the assembly, nuclear retention, activity, and processivity of the complex (Mollenbeck et al 2003;Aigner and Cech 2004;Witkin and Collins 2004). P65 is one of the first factors binding to telomerase RNA.…”

Section: Introductionmentioning

confidence: 99%

A comprehensive analysis of the La-motif protein superfamily

Bousquet‐Antonelli¹,

Deragon²

2009

RNA

166

284

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“…These results reveal that the p65-TER complex recruits TERT in strong preference to TERT association with TER alone. After these experiments were completed, a ten-fold excess of T. thermophila TER was shown to reduce the association of E. aediculatus p43 with T. thermophila TERT 24 . Tetrahymena thermophila p65 and E. aediculatus p43 have limited sequence conservation beyond their shared homology with La proteins, differing even in the predicted consensus residues of the p65 RRM 9 ; their cognate TER stem IV regions share no primary sequence conservation and have major differences in stem lengths, bulges and potential branches 25 .…”

Section: Assembly Of a P65-ter-tert Complexmentioning

confidence: 99%

A telomerase holoenzyme protein enhances telomerase RNA assembly with telomerase reverse transcriptase

Prathapam

Witkin

O’Connor

et al. 2005

Nat Struct Mol Biol

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Telomerase maintains the simple sequence repeats at chromosome ends, protecting cells from genomic rearrangement, proliferative senescence and death. The telomerase reverse transcriptase (TERT) and telomerase RNA (TER) alone can assemble into active enzyme in a heterologous cell extract, but the physiological process of telomerase biogenesis is more complex. The endogenous accumulation of Tetrahymena thermophila TERT and TER requires an additional telomerase holoenzyme protein, p65. Here, we reconstitute this cellular pathway for telomerase ribonucleoprotein biogenesis in vitro. We demonstrate that tandem RNA interaction domains in p65 recognize the sequence of the TER 3′ stem. Notably, the p65-TER complex recruits TERT much more efficiently than does TER alone. Using bacterially expressed p65 and TERT polypeptides, we show that p65 enhances TERT-TER interaction by a mechanism involving a conserved bulge in the protein-bridging TER molecule. These findings reveal a pathway for telomerase holoenzyme biogenesis that preassembles TER for TERT recruitment.Cellular ribonucleoprotein (RNP) biogenesis has gained attention as a dynamic, regulated cascade of events that can be affected in human disease 1-3. RNPs assemble in vivo aided both by assembly chaperones, which contribute transient interactions, and by architectural RNA-binding proteins, which remain integral components of an assembled RNP 4 . Progress in the elucidation of physiological RNP biogenesis mechanisms has been impeded by the limited success in reconstitution of ordered protein and RNA assembly pathways in vitro.TERT and TER can assemble in rabbit reticulocyte lysate (RRL) to form a minimal recombinant telomerase RNP 5 . The physiological process of telomerase enzyme biogenesis, however, seems more complex. In vivo, human TER accumulation and telomere length maintenance are compromised by disease-associated single-residue substitutions in the RNA-binding protein dyskerin 6,7 . In budding yeast, TER accumulation requires RNAbinding proteins from the Sm family 8 . The potentially pleiotropic impact of vertebrate dyskerin or yeast Sm protein alterations complicates the interpretation of their direct roles in telomerase RNP assembly. Additionally, studies of these RNP assembly pathways in vitro are hampered by the inability to reconstitute a dyskerin or Sm RNP efficiently in the absence of other, typically concerted cellular events. In the biological context, it was thus unresolved whether TERT assembles with TER unassisted or only transiently chaperoned, or whether [9][10][11][12] . Genetic depletion of T. thermophila p65 reduces TER accumulation without impact on other small nuclear RNAs, suggesting that in contrast to vertebrate dyskerin or yeast Sm proteins, p65 has a telomerase-specific function in RNP biogenesis 9 . Based on these and additional findings, we proposed a p65-dependent pathway for the physiological biogenesis of T. thermophila telomerase RNP. Here, we test the proposed telomerase RNP assembly pathway by reconstituting the inter...

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“…Structural and biochemical studies (Alfano et al 2003(Alfano et al , 2004Dong et al 2004) on the genuine La protein (LARP3) revealed specific binding of the La module to 3 ′ oligo-uridylic acid stretches (Stefano 1984;Wolin and Cedervall 2002;Teplova et al 2006;KotikKogan et al 2008). Accordingly, members of the LARP3 and LARP7 families associate predominantly with RNA polymerase III (RNAPIII) transcripts in vivo and influence their stability and/or maturation pathways (Sobel and Wolin 1999;Intine et al 2000;Aigner et al 2003;Chakshusmathi et al 2003;Aigner and Cech 2004;Prathapam et al 2005;Stone et al 2007;Copela et al 2008;Krueger et al 2008;Markert et al 2008). While other LARP family members also contain La modules, recent evidence suggested that LARP1, LARP4, and LARP6 are associated with mRNAs rather than RNAPIII transcripts (Nykamp et al 2008;Blagden et al 2009;Wang et al 2009;Weng et al 2009;Burrows et al 2010;Cai et al 2010a;Schäffler et al 2010;Zanin et al 2010;Yang et al 2011;Aoki et al 2013;Mura et al 2014;Tcherkezian et al 2014).…”

Section: Introductionmentioning

confidence: 99%

LARP4B is an AU-rich sequence associated factor that promotes mRNA accumulation and translation

Küspert

Murakawa

Schäffler

et al. 2015

RNA

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mRNAs are key molecules in gene expression and subject to diverse regulatory events. Regulation is accomplished by distinct sets of trans-acting factors that interact with mRNAs and form defined mRNA-protein complexes (mRNPs). The resulting "mRNP code" determines the fate of any given mRNA and thus controlling gene expression at the post-transcriptional level. The Larelated protein 4B (LARP4B) belongs to an evolutionarily conserved family of RNA-binding proteins characterized by the presence of a La-module implicated in direct RNA binding. Biochemical experiments have shown previously direct interactions of LARP4B with factors of the translation machinery. This finding along with the observation of an association with actively translating ribosomes suggested that LARP4B is a factor contributing to the mRNP code. To gain insight into the function of LARP4B in vivo we tested its mRNA association at the transcriptome level and its impact on the proteome. PAR-CLIP analyses allowed us to identify the in vivo RNA targets of LARP4B. We show that LARP4B binds to a distinct set of cellular mRNAs by contacting their 3 ′ UTRs. Biocomputational analysis combined with in vitro binding assays identified the LARP4B-binding motif on mRNA targets. The reduction of cellular LARP4B levels leads to a marked destabilization of its mRNA targets and consequently their reduced translation. Our data identify LARP4B as a component of the mRNP code that influences the expression of its mRNA targets by affecting their stability.

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The Euplotes telomerase subunit p43 stimulates enzymatic activity and processivity in vitro

Cited by 27 publications

References 52 publications

A comprehensive analysis of the La-motif protein superfamily

A comprehensive analysis of the La-motif protein superfamily

A telomerase holoenzyme protein enhances telomerase RNA assembly with telomerase reverse transcriptase

LARP4B is an AU-rich sequence associated factor that promotes mRNA accumulation and translation

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