ABSTRACT
Many small nucleolar RNAs (snoRNA)s are processed from introns of host genes, but the importance of splicing for proper biogenesis and the fate of the snoRNAs is not well understood. Here, we show that inactivation of splicing factors or mutation of splicing signals leads to the accumulation of partially processed hybrid messenger RNA-snoRNA (hmsnoRNA) transcripts. hmsnoRNAs are processed to the mature 3' ends of the snoRNAs by the nuclear exosome and bound by small nucleolar ribonucleoproteins. hmsnoRNAs are unaffected by translation-coupled RNA quality-control pathways, but they are degraded by the major cytoplasmic exonuclease Xrn1p, due to their messenger RNA (mRNA)-like 5' extensions. These results show that completion of splicing is required to promote complete and accurate processing of intron-encoded snoRNAs and that splicing defects lead to degradation of hybrid mRNA-snoRNA species by cytoplasmic decay, underscoring the importance of splicing for the biogenesis of intron-encoded snoRNAs.
Subject(s)
RNA Splicing , RNA Stability , RNA, Messenger , RNA, Small Nucleolar , Introns , RNA, Messenger/genetics , RNA, Small Nucleolar/geneticsABSTRACT
The N6-methyladenosine modification (m6A) modulates eukaryotic mRNA decay. In this issue of Cell Reports, Boo et al. describe a mechanism for degradation of m6A-containing mRNAs by 5'-decapping, which occurs through the recruitment of the degradation factor UPF1 via the m6A reader protein YTHDF2.
Subject(s)
Adenosine , RNA-Binding Proteins , Adenosine/metabolism , Animals , Feathers/metabolism , RNA Stability/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolismABSTRACT
BipA is a conserved translational GTPase of bacteria recently implicated in ribosome biogenesis. Here we show that Escherichia coli ΔbipA cells grown at suboptimal temperature accumulate immature large subunit particles missing several proteins. These include L17 and L17-dependent binders, suggesting that structural block 3 of the subunit folds late in the assembly process. Parallel analysis of the control strain revealed accumulation of nearly identical intermediates, albeit at lower levels, suggesting qualitatively similar routes of assembly. This came as a surprise, because earlier analogous studies of wild-type E. coli showed early binding of L17. Further investigation showed that the main path of 50S assembly differs depending on conditions of growth. Either supplementation of the media with lysine and arginine or suboptimal temperature appears to delay block 3 folding, demonstrating the flexible nature of the assembly process. We also show that the variant BipA-H78A fails to rescue phenotypes of the ΔbipA strain, indicating a critical role for GTP hydrolysis in BipA function. In fact, BipA-H78A confers a dominant negative phenotype in wild-type cells. Controlled production of BipA-H78A causes accumulation of 70S monosomes at the expense of polysomes, suggesting that the growth defect stems from a shutdown of translation.
Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , GTP Phosphohydrolases/metabolism , Ribosome Subunits, Large, Bacterial/metabolism , Escherichia coli/genetics , Escherichia coli/growth & development , Escherichia coli Proteins/genetics , GTP Phosphohydrolases/genetics , Guanosine Triphosphate/metabolism , Hydrolysis , Models, Molecular , Mutation , Protein Biosynthesis , Ribosome Subunits, Large, Bacterial/geneticsABSTRACT
Protein synthesis relies on several translational GTPases (trGTPases), related proteins that couple the hydrolysis of GTP to specific molecular events on the ribosome. Most bacterial trGTPases, including IF2, EF-Tu, EF-G and RF3, play well-known roles in translation. The cellular functions of LepA (also termed EF4) and BipA (also termed TypA), conversely, have remained enigmatic. Recent studies provide compelling in vivo evidence that LepA and BipA function in biogenesis of the 30S and 50S subunit respectively. These findings have important implications for ribosome biogenesis in bacteria. Because the GTPase activity of each of these proteins depends on interactions with both ribosomal subunits, some portion of 30S and 50S assembly must occur in the context of the 70S ribosome. In this review, we introduce the trGTPases of bacteria, describe the new functional data on LepA and BipA, and discuss the how these findings shape our current view of ribosome biogenesis in bacteria.
Subject(s)
Bacteria/enzymology , GTP Phosphohydrolases/metabolism , Ribosome Subunits, Large, Bacterial/metabolism , Ribosome Subunits, Small, Bacterial/metabolism , GTP Phosphohydrolases/genetics , Peptide Elongation Factors/genetics , Peptide Elongation Factors/metabolism , Peptide Initiation Factors/genetics , Peptide Initiation Factors/metabolism , Phylogeny , Protein BiosynthesisABSTRACT
The physiological role of LepA, a paralog of EF-G found in all bacteria, has been a mystery for decades. Here, we show that LepA functions in ribosome biogenesis. In cells lacking LepA, immature 30S particles accumulate. Four proteins are specifically underrepresented in these particles-S3, S10, S14, and S21-all of which bind late in the assembly process and contribute to the folding of the 3' domain of 16S rRNA. Processing of 16S rRNA is also delayed in the mutant strain, as indicated by increased levels of precursor 17S rRNA in assembly intermediates. Mutation ΔlepA confers a synthetic growth phenotype in absence of RsgA, another GTPase, well known to act in 30S subunit assembly. Analysis of the ΔrsgA strain reveals accumulation of intermediates that resemble those seen in the absence of LepA. These data suggest that RsgA and LepA play partially redundant roles to ensure efficient 30S assembly.
