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RNA Biology Feb 2021La-related proteins (LARPs) share a La motif (LaM) followed by an RNA recognition motif (RRM). Together these are termed the La-module that, in the prototypical nuclear... (Review)
Review
La-related proteins (LARPs) share a La motif (LaM) followed by an RNA recognition motif (RRM). Together these are termed the La-module that, in the prototypical nuclear La protein and LARP7, mediates binding to the UUU-3'OH termination motif of nascent RNA polymerase III transcripts. We briefly review La and LARP7 activities for RNA 3' end binding and protection from exonucleases before moving to the more recently uncovered poly(A)-related activities of LARP1 and LARP4. Two features shared by LARP1 and LARP4 are direct binding to poly(A) and to the cytoplasmic poly(A)-binding protein (PABP, also known as PABPC1). LARP1, LARP4 and other proteins involved in mRNA translation, deadenylation, and decay, contain PAM2 motifs with variable affinities for the MLLE domain of PABP. We discuss a model in which these PABP-interacting activities contribute to poly(A) pruning of active mRNPs. Evidence that the SARS-CoV-2 RNA virus targets PABP, LARP1, LARP 4 and LARP 4B to control mRNP activity is also briefly reviewed. Recent data suggests that LARP4 opposes deadenylation by stabilizing PABP on mRNA poly(A) tails. Other data suggest that LARP1 can protect mRNA from deadenylation. This is dependent on a PAM2 motif with unique characteristics present in its La-module. Thus, while nuclear La and LARP7 stabilize small RNAs with 3' oligo(U) from decay, LARP1 and LARP4 bind and protect mRNA 3' poly(A) tails from deadenylases through close contact with PABP.: 5'TOP: 5' terminal oligopyrimidine, LaM: La motif, LARP: La-related protein, LARP1: La-related protein 1, MLLE: mademoiselle, NTR: N-terminal region, PABP: cytoplasmic poly(A)-binding protein (PABPC1), Pol III: RNA polymerase III, PAM2: PABP-interacting motif 2, PB: processing body, RRM: RNA recognition motif, SG: stress granule.
Topics: Amino Acid Motifs; Autoantigens; Humans; Phylogeny; Poly A; Poly(A)-Binding Proteins; Protein Binding; Protein Biosynthesis; Protein Domains; RNA Stability; RNA, Messenger; RNA, Viral; Ribonucleoproteins; SARS-CoV-2; SS-B Antigen
PubMed: 33522422
DOI: 10.1080/15476286.2020.1868753 -
Development (Cambridge, England) Jun 2022As one of the post-transcriptional regulatory mechanisms, uncoupling of transcription and translation plays an essential role in development and adulthood physiology....
As one of the post-transcriptional regulatory mechanisms, uncoupling of transcription and translation plays an essential role in development and adulthood physiology. However, it remains elusive how thousands of mRNAs get translationally silenced while stability is maintained for hours or even days before translation. In addition to oocytes and neurons, developing spermatids display significant uncoupling of transcription and translation for delayed translation. Therefore, spermiogenesis represents an excellent in vivo model for investigating the mechanism underlying uncoupled transcription and translation. Through full-length poly(A) deep sequencing, we discovered dynamic changes in poly(A) length through deadenylation and re-polyadenylation. Deadenylation appeared to be mediated by microRNAs (miRNAs), and transcripts with shorter poly(A) tails tend to be sequestered into ribonucleoprotein (RNP) granules for translational repression and stabilization. In contrast, re-polyadenylation might allow for translocation of the translationally repressed transcripts from RNP granules to polysomes. Overall, our data suggest that miRNA-dependent poly(A) length control represents a previously unreported mechanism underlying uncoupled translation and transcription in haploid male mouse germ cells.
Topics: Animals; Haploidy; Male; Mice; MicroRNAs; Poly A; Protein Biosynthesis; RNA, Messenger; Spermatids
PubMed: 35588208
DOI: 10.1242/dev.199573 -
Experimental & Molecular Medicine Feb 2023Translation is mediated by precisely orchestrated sequential interactions among translation initiation components, mRNA, and ribosomes. Biochemical, structural, and... (Review)
Review
Translation is mediated by precisely orchestrated sequential interactions among translation initiation components, mRNA, and ribosomes. Biochemical, structural, and genetic techniques have revealed the fundamental mechanism that determines what occurs and when, where and in what order. Most mRNAs are circularized via the eIF4E-eIF4G-PABP interaction, which stabilizes mRNAs and enhances translation by recycling ribosomes. However, studies using single-molecule fluorescence imaging have allowed for the visualization of complex data that opposes the traditional "functional circularization" theory. Here, we briefly introduce single-molecule techniques applied to studies on mRNA circularization and describe the results of in vitro and live-cell imaging. Finally, we discuss relevant insights and questions gained from single-molecule research related to translation.
Topics: RNA, Messenger; Protein Biosynthesis; Poly(A)-Binding Proteins; Protein Binding; Eukaryotic Initiation Factor-4G
PubMed: 36720916
DOI: 10.1038/s12276-023-00933-1 -
Chemistry (Weinheim An Der Bergstrasse,... Jul 2022Poly(A)-binding protein (PABP) is an essential element of cellular translational machinery. Recent studies have revealed that poly(A) tail modifications can modulate...
Poly(A)-binding protein (PABP) is an essential element of cellular translational machinery. Recent studies have revealed that poly(A) tail modifications can modulate mRNA stability and translational potential, and that oligoadenylate-derived PABP ligands can act as effective translational inhibitors with potential applications in pain management. Although extensive research has focused on protein-RNA and protein-protein interactions involving PABPs, further studies are required to examine the ligand specificity of PABP. In this study, we developed a microscale thermophoresis-based assay to probe the interactions between PABP and oligoadenylate analogs containing different chemical modifications. Using this method, we evaluated oligoadenylate analogs modified with nucleobase, ribose, and phosphate moieties to identify modification hotspots. In addition, we determined the susceptibility of the modified oligos to CNOT7 to identify those with the potential for increased cellular stability. Consequently, we selected two enzymatically stable oligoadenylate analogs that inhibit translation in rabbit reticulocyte lysates with a higher potency than a previously reported PABP ligand. We believe that the results presented in this study and the implemented methodology can be capitalized upon in the future development of RNA-based biological tools.
Topics: Animals; Ligands; Poly A; Poly(A)-Binding Proteins; Protein Binding; Protein Biosynthesis; RNA; RNA, Messenger; Rabbits; Structure-Activity Relationship
PubMed: 35575378
DOI: 10.1002/chem.202201115 -
Methods in Molecular Biology (Clifton,... 2021Ribosome profiling is a powerful technique that enables researchers to monitor translational events across the transcriptome. It provides a snapshot of ribosome... (Comparative Study)
Comparative Study
Ribosome profiling is a powerful technique that enables researchers to monitor translational events across the transcriptome. It provides a snapshot of ribosome positions and density across the transcriptome at a sub-codon resolution. Here we describe the whole procedure of profiling ribosome footprints in mammalian cells. Two methods for Ribo-seq library construction are introduced, and their advantages and disadvantages are compared. There is a room for further improvement of Ribo-seq in terms of the amount of starting material, the duration of library construction, and the resolution of sequencing results.
Topics: Gene Library; HEK293 Cells; High-Throughput Nucleotide Sequencing; Humans; Poly A; Protein Biosynthesis; RNA, Messenger; Ribosomes; Sequence Analysis, RNA; Software
PubMed: 33765278
DOI: 10.1007/978-1-0716-1150-0_10 -
Cells Feb 2023In eukaryotes, mRNA metabolism requires a sophisticated signaling system. Recent studies have suggested that polyadenylate tail may play a vital role in such a system....
In eukaryotes, mRNA metabolism requires a sophisticated signaling system. Recent studies have suggested that polyadenylate tail may play a vital role in such a system. The poly(A) tail used to be regarded as a common modification at the 3' end of mRNA, but it is now known to be more than just that. It appears to act as a platform or hub that can be understood in two ways. On the one hand, polyadenylation and deadenylation machinery constantly regulates its dynamic activity; on the other hand, it exhibits the ability to recruit RNA-binding proteins and then interact with diverse factors to send various signals to regulate mRNA metabolism. In this paper, we outline the main complexes that regulate the dynamic activities of poly(A) tails, explain how these complexes participate polyadenylation/deadenylation process and summarize the diverse signals this hub emit. We are trying to make a point that the poly(A) tail can metaphorically act as a "flagman" who is supervised by polyadenylation and deadenylation and sends out signals to regulate the orderly functioning of mRNA metabolism.
Topics: Polyadenylation; RNA-Binding Proteins; RNA, Messenger
PubMed: 36831239
DOI: 10.3390/cells12040572 -
The Journal of Biological Chemistry Aug 2023Poly(A)-binding protein nuclear 1 (PABPN1) is an RNA-binding protein localized in nuclear speckles, while its alanine (Ala)-expanded variants accumulate as intranuclear...
Poly(A)-binding protein nuclear 1 (PABPN1) is an RNA-binding protein localized in nuclear speckles, while its alanine (Ala)-expanded variants accumulate as intranuclear aggregates in oculopharyngeal muscular dystrophy. The factors that drive PABPN1 aggregation and its cellular consequences remain largely unknown. Here, we investigated the roles of Ala stretch and poly(A) RNA in the phase transition of PABPN1 using biochemical and molecular cell biology methods. We have revealed that the Ala stretch controls its mobility in nuclear speckles, and Ala expansion leads to aggregation from the dynamic speckles. Poly(A) nucleotide is essential to the early-stage condensation that thereby facilitates speckle formation and transition to solid-like aggregates. Moreover, the PABPN1 aggregates can sequester CFIm25, a component of the pre-mRNA 3'-UTR processing complex, in an mRNA-dependent manner and consequently impair the function of CFIm25 in alternative polyadenylation. In conclusion, our study elucidates a molecular mechanism underlying PABPN1 aggregation and sequestration, which will be beneficial for understanding PABPN1 proteinopathy.
Topics: Humans; Alanine; Muscular Dystrophy, Oculopharyngeal; Poly(A)-Binding Protein I; Polyadenylation; RNA
PubMed: 37422193
DOI: 10.1016/j.jbc.2023.105019 -
Cell Reports Oct 2022Translation of 5' terminal oligopyrimidine (TOP) mRNAs encoding the protein synthesis machinery is strictly regulated by an amino-acid-sensing mTOR pathway. However, its...
Translation of 5' terminal oligopyrimidine (TOP) mRNAs encoding the protein synthesis machinery is strictly regulated by an amino-acid-sensing mTOR pathway. However, its regulatory mechanism remains elusive. Here, we demonstrate that TOP mRNA translation positively correlates with its poly(A) tail length under mTOR active/amino-acid-rich conditions, suggesting that TOP mRNAs are post-transcriptionally controlled by poly(A) tail-length regulation. Consistent with this, the tail length of TOP mRNAs dynamically fluctuates in response to amino acid availability. The poly(A) tail shortens under mTOR active/amino-acid-rich conditions, whereas the long-tailed TOP mRNAs accumulate under mTOR inactive/amino-acid-starved (AAS) conditions. An RNA-binding protein, LARP1, is indispensable for the process. LARP1 interacts with non-canonical poly(A) polymerases and induces post-transcriptional polyadenylation of the target. Our findings illustrate that LARP1 contributes to the selective accumulation of TOP mRNAs with long poly(A) tails under AAS, resulting in accelerated ribosomal loading onto TOP mRNAs for the resumption of translation after AAS.
Topics: RNA, Messenger; Ribonucleoproteins; Autoantigens; TOR Serine-Threonine Kinases; Ribosomes; RNA-Binding Proteins; Polynucleotide Adenylyltransferase; Amino Acids; Protein Biosynthesis
PubMed: 36288708
DOI: 10.1016/j.celrep.2022.111548 -
Methods in Molecular Biology (Clifton,... 2022Polyadenylation and deadenylation of mRNA are major RNA modifications associated with nucleus-to-cytoplasm translocation, mRNA stability, translation efficiency, and...
Polyadenylation and deadenylation of mRNA are major RNA modifications associated with nucleus-to-cytoplasm translocation, mRNA stability, translation efficiency, and mRNA decay pathways. Our current knowledge of polyadenylation and deadenylation has been expanded due to recent advances in transcriptome-wide poly(A) tail length assays. Whereas these methods measure poly(A) length by quantifying the adenine (A) base stretch at the 3' end of mRNA, we developed a more cost-efficient technique that does not rely on A-base counting, called tail-end-displacement sequencing (TED-seq). Through sequencing highly size-selected 3' RNA fragments including the poly(A) tail pieces, TED-seq provides accurate measure of transcriptome-wide poly(A)-tail lengths in high resolution, economically suitable for larger scale analysis under various biologically transitional contexts.
Topics: Genome; Poly A; Polyadenylation; RNA Stability; RNA, Messenger; Sequence Analysis, RNA
PubMed: 34694615
DOI: 10.1007/978-1-0716-1851-6_15 -
STAR Protocols May 2023Poly(A) tail metabolism contributes to post-transcriptional regulation of gene expression. Here, we present a protocol for analyzing intact mRNA poly(A) tail length...
Poly(A) tail metabolism contributes to post-transcriptional regulation of gene expression. Here, we present a protocol for analyzing intact mRNA poly(A) tail length using nanopore direct RNA sequencing, which excludes truncated RNAs from the measurement. We describe steps for preparing recombinant eIF4E mutant protein, purifying m7G- capped RNAs, library preparation, and sequencing. Resulting data can be used not only for expression profiling and poly(A) tail length estimation but also for detecting alternative splicing and polyadenylation events and RNA base modification. For complete details on the use and execution of this protocol, please refer to Ogami et al. (2022)..
PubMed: 37243600
DOI: 10.1016/j.xpro.2023.102340