Estimation of Myostatin gene effects on production traits and fatty acid contents in bovine milk

peer reviewedThe aim of this study was to estimate the genetic parameters of milk, fat, and protein yields, saturated (SFA) and monounsaturated fatty acid (MUFA) contents in bovine milk and to estimate the Myostatin (mh) gene effect on these traits. For this purpose, 51,614 test-day records (24,124, 16,145, and 11,345 for first, second and third lactation, respectively) of 3,098 dual purpose Belgian Blue cows in 38 herds from the Walloon Region of Belgium were used. Because only 2,301 animals, including 1,082 cows with test-day records, were genotyped for mh, the gene content of non-genotyped animals was predicted from animals with a known genotype using the relationships with these animals. Variance components were estimated using Restricted Maximum Likelihood. A 3-lactations, 5-traits random regression test-day mixed model, based on the official Walloon genetic evaluation model for production traits, was used with an additional fixed regression on mh gene content to estimate allele substitution effects. Daily heritability estimates (average of 3 lactations) were 0.34 for SFA and 0.16 for MUFA and were higher than those for production traits (0.11, 0.10, and 0.09 for milk, fat, and protein yields, respectively). Allele substitution effects approximate standard-errors) for mh through the three lactations were-0.628 (+0.343),-0.024 (0.014) and -0.021 (+0.009) kg per day for milk, fat, and protein yields, respectively. Concerning SFA and MUFA contents in milk, the average allele substitution effects were -0.001 (+0.027) and 0.029 (+0.023) g/dl of milk. To conclude, results from this study showed that milk performance traits and milk fatty acid profile are influenced by mh genotypes

Rio1 mediates ATP-dependent final maturation of 40S ribosomal subunits

During the last step in 40S ribosome subunit biogen-esis, the PIN-domain endonuclease Nob1 cleaves the 20S pre-rRNA at site D, to form the mature 18S rRNAs. Here we report that cleavage occurs in particles that have largely been stripped of previously character-ized pre-40S components, but retain the endonu-clease Nob1, its binding partner Pno1 (Dim2) and the atypical ATPase Rio1. Within the Rio1-associated pre-40S particles, in vitro pre-rRNA cleavage was strongly stimulated by ATP and required nucleotide binding by Rio1. In vivo binding sites for Rio1, Pno1 and Nob1 were mapped by UV cross-linking in ac-tively growing cells. Nob1 and Pno1 bind overlap-ping regions within the internal transcribed spacer 1, and both bind directly over cleavage site D. Bind-ing sites for Rio1 were within the core of the 18S rRNA, overlapping tRNA interaction sites and distinct from the related kinase Rio2. Site D cleavage occurs within pre-40S-60S complexes and Rio1-associated particles efficiently assemble into these complexes, whereas Pno1 appeared to be depleted relative to Nob1. We speculate that Rio1-mediated dissociation of Pno1 from cleavage site D is the trigger for final 18S rRNA maturation

Distinct cytoplasmic maturation steps of 40S ribosomal subunit precursors require hRio2

During their biogenesis, 40S ribosomal subunit precursors are exported from the nucleus to the cytoplasm, where final maturation occurs. In this study, we show that the protein kinase human Rio2 (hRio2) is part of a late 40S preribosomal particle in human cells. Using a novel 40S biogenesis and export assay, we analyzed the contribution of hRio2 to late 40S maturation. Although hRio2 is not absolutely required for pre-40S export, deletion of its binding site for the export receptor CRM1 decelerated the kinetics of this process. Moreover, in the absence of hRio2, final cytoplasmic 40S maturation is blocked because the recycling of several trans-acting factors and cytoplasmic 18S-E precursor ribosomal RNA (rRNA [pre-rRNA]) processing are defective. Intriguingly, the physical presence of hRio2 but not its kinase activity is necessary for the release of hEnp1 from cytoplasmic 40S precursors. In contrast, hRio2 kinase activity is essential for the recycling of hDim2, hLtv1, and hNob1 as well as for 18S-E pre-rRNA processing. Thus, hRio2 is involved in late 40S maturation at several distinct steps

An evolutionary intra-molecular shift in the preferred U3 snoRNA binding site on pre-ribosomal RNA

Correct docking of U3 small nucleolar RNA (snoRNA) on pre-ribosomal RNA (pre-rRNA) is essential for rRNA processing to produce 18S rRNA. In this report, we have used Xenopus oocytes to characterize the structural requirements of the U3 snoRNA 3′-hinge interaction with region E1 of the external transcribed spacer (ETS) of pre-rRNA. This interaction is crucial for docking to initiate rRNA processing. 18S rRNA production was inhibited when fewer than 6 of the 8 bp of the U3 3′–hinge complex with the ETS could form; moreover, base pairing involving the right side of the 3′-hinge was more important than the left. Increasing the length of the U3 hinge–ETS interaction by 9 bp impaired rRNA processing. Formation of 18S rRNA was also inhibited by swapping the U3 5′- and 3′-hinge interactions with the ETS or by shifting the base pairing of the U3 3′-hinge to the sequence directly adjacent to ETS region E1. However, 18S rRNA production was partially restored by a compensatory shift that allowed the sequence adjacent to the U3 3′-hinge to pair with the eight bases directly adjacent to ETS region E1. The results suggest that the geometry of the U3 snoRNA interaction with the ETS is critical for rRNA processing

Analysis of two human pre-ribosomal factors, bystin and hTsr1, highlights differences in evolution of ribosome biogenesis between yeast and mammals

Recent studies reveal that maturation of the 40S ribosomal subunit precursors in mammals includes an additional step during processing of the internal transcribed spacer 1 (ITS1), when compared with yeast Saccharomyces cerevisiae, even though the protein content of the pre-40S particle appears to be the same. Here, we examine by depletion with siRNA treatment the function of human orthologs of two essential yeast pre-ribosomal factors, hEnp1/bystin and hTsr1. Like their yeast orthologs, bystin is required for efficient cleavage of the ITS1 and further processing of this domain within the pre-40S particles, whereas hTsr1 is necessary for the final maturation steps. However, bystin depletion leads to accumulation of an unusual 18S rRNA precursor, revealing a new step in ITS1 processing that potentially involves an exonuclease. In addition, pre-40S particles lacking hTsr1 are partially retained in the nucleus, whereas depletion of Tsr1p in yeast results in strong cytoplasmic accumulation of pre-40S particles. These data indicate that ITS1 processing in human cells may be more complex than currently envisioned and that coordination between maturation and nuclear export of pre-40S particles has evolved differently in yeast and mammalian cells

Cryo-EM structure of a late pre-40S ribosomal subunit from Saccharomyces cerevisiae

Mechanistic understanding of eukaryotic ribosome formation requires a detailed structural knowledge of the numerous assembly intermediates, generated along a complex pathway. Here, we present the structure of a late pre-40S particle at 3.6 Å resolution, revealing in molecular detail how assembly factors regulate the timely folding of pre-18S rRNA. The structure shows that, rather than sterically blocking 40S translational active sites, the associated assembly factors Tsr1, Enp1, Rio2 and Pno1 collectively preclude their final maturation, thereby preventing untimely tRNA and mRNA binding and error prone translation. Moreover, the structure explains how Pno1 coordinates the 3'end cleavage of the 18S rRNA by Nob1 and how the late factor's removal in the cytoplasm ensures the structural integrity of the maturing 40S subunit

A novel family of plant nuclear envelope associated proteins

This paper describes the characterisation of a new family of higher plant nuclear envelope associated proteins (NEAPs) that interact with proteins of the nuclear envelope. In the model plant Arabidopsis thaliana, the family consists of three genes expressed ubiquitously (AtNEAP1-3) and a pseudogene (AtNEAP4). NEAPs consist of extensive coiled-coil domains, followed by a nuclear localisation signal and a C-terminal predicted transmembrane domain. Domain deletion mutants confirm the presence of a functional nuclear localisation signal and transmembrane domain. AtNEAP proteins localise to the nuclear periphery as part of stable protein complexes, are able to form homo- and heteromers and interact with the SUN domain proteins AtSUN1 and AtSUN2, involved in the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. An A. thaliana cDNA library screen identified a putative transcription factor called AtbZIP18 as a novel interactor of AtNEAP1, which suggest a connection between NEAP and chromatin. An Atneap1 Atneap3 double knock out mutant showed reduced root growth and altered nuclear morphology and chromatin structure. Thus AtNEAPs are suggested as INM anchored coiled-coil proteins with roles in maintaining nuclear morphology and chromatin structure

Estimation of myostatin gene effects on production traits and fatty acid contents in bovine milk

peer reviewedThe aim of this study was to estimate the genetic parameters of milk, fat, and protein yields, saturated (SFA) and monounsaturated fatty acid (MUFA) contents in bovine milk and to estimate the Myostatin (mh) gene effect on these traits. For this purpose, 51,614 test-day records (24,124, 16,145, and 11,345 for first, second and third lactation, respectively) of 3,098 dual purpose Belgian Blue cows in 38 herds from the Walloon Region of Belgium were used. Because only 2,301 animals, including 1,082 cows with test-day records, were genotyped for mh, the gene content of non-genotyped animals was predicted from animals with a known genotype using the relationships with these animals. Variance components were estimated using Restricted Maximum Likelihood. A 3-lactations, 5-traits random regression test-day mixed model, based on the official Walloon genetic evaluation model for production traits, was used with an additional fixed regression on mh gene content to estimate allele substitution effects. Daily heritability estimates (average of 3 lactations) were 0.34 for SFA and 0.16 for MUFA and were higher than those for production traits (0.11, 0.10, and 0.09 for milk, fat, and protein yields, respectively). Allele substitution effects approximate standard-errors) for mh through the three lactations were-0.628 (+0.343),-0.024 (0.014) and -0.021 (+0.009) kg per day for milk, fat, and protein yields, respectively. Concerning SFA and MUFA contents in milk, the average allele substitution effects were -0.001 (+0.027) and 0.029 (+0.023) g/dl of milk. To conclude, results from this study showed that milk performance traits and milk fatty acid profile are influenced by mh genotypes

Protein-Protein Interactions within Late Pre-40S Ribosomes

Ribosome assembly in eukaryotic organisms requires more than 200 assembly factors to facilitate and coordinate rRNA transcription, processing, and folding with the binding of the ribosomal proteins. Many of these assembly factors bind and dissociate at defined times giving rise to discrete assembly intermediates, some of which have been partially characterized with regards to their protein and RNA composition. Here, we have analyzed the protein-protein interactions between the seven assembly factors bound to late cytoplasmic pre-40S ribosomes using recombinant proteins in binding assays. Our data show that these factors form two modules: one comprising Enp1 and the export adaptor Ltv1 near the beak structure, and the second comprising the kinase Rio2, the nuclease Nob1, and a regulatory RNA binding protein Dim2/Pno1 on the front of the head. The GTPase-like Tsr1 and the universally conserved methylase Dim1 are also peripherally connected to this second module. Additionally, in an effort to further define the locations for these essential proteins, we have analyzed the interactions between these assembly factors and six ribosomal proteins: Rps0, Rps3, Rps5, Rps14, Rps15 and Rps29. Together, these results and previous RNA-protein crosslinking data allow us to propose a model for the binding sites of these seven assembly factors. Furthermore, our data show that the essential kinase Rio2 is located at the center of the pre-ribosomal particle and interacts, directly or indirectly, with every other assembly factor, as well as three ribosomal proteins required for cytoplasmic 40S maturation. These data suggest that Rio2 could play a central role in regulating cytoplasmic maturation steps

Analysis of ribosome biogenesis factor-modules in yeast cells depleted from pre-ribosomes

Formation of eukaryotic ribosomes requires more than 150 biogenesis factors which transiently interact with the nascent ribosomal subunits. Previously, many pre-ribosomal intermediates could be distinguished by their protein composition and rRNA precursor (pre-rRNA) content. We purified complexes of ribosome biogenesis factors from yeast cells in which de novo synthesis of rRNA precursors was down-regulated by genetic means. We compared the protein composition of these largely pre-rRNA free assemblies with the one of analogous pre-ribosomal preparations by semi-quantitative mass spectrometry. The experimental setup minimizes the possibility that the analysed pre-rRNA free protein modules were derived from (partially) disrupted pre-ribosomal particles and provides thereby strong evidence for their pre-ribosome independent existence. In support of the validity of this approach (i) the predicted composition of the analysed protein modules was in agreement with previously described rRNA-free complexes and (ii) in most of the cases we could identify new candidate members of reported protein modules. An unexpected outcome of these analyses was that free large ribosomal subunits are associated with a specific set of ribosome biogenesis factors in cells where neo-production of nascent ribosomes was blocked. The data presented strengthen the idea that assembly of eukaryotic pre-ribosomal particles can result from transient association of distinct building blocks