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FEBS Open Bio Jul 2023During their synthesis in the cell nucleus, most eukaryotic mRNAs undergo a two-step 3'-end processing reaction in which the pre-mRNA is cleaved and released from the... (Review)
Review
During their synthesis in the cell nucleus, most eukaryotic mRNAs undergo a two-step 3'-end processing reaction in which the pre-mRNA is cleaved and released from the transcribing RNA polymerase II and a polyadenosine (poly(A)) tail is added to the newly formed 3'-end. These biochemical reactions might appear simple at first sight (endonucleolytic RNA cleavage and synthesis of a homopolymeric tail), but their catalysis requires a multi-faceted enzymatic machinery, the cleavage and polyadenylation complex (CPAC), which is composed of more than 20 individual protein subunits. The activity of CPAC is further orchestrated by Poly(A) Binding Proteins (PABPs), which decorate the poly(A) tail during its synthesis and guide the mRNA through subsequent gene expression steps. Here, we review the structure, molecular mechanism, and regulation of eukaryotic mRNA 3'-end processing machineries with a focus on the polyadenylation step. We concentrate on the CPAC and PABPs from mammals and the budding yeast, Saccharomyces cerevisiae, because these systems are the best-characterized at present. Comparison of their functions provides valuable insights into the principles of mRNA 3'-end processing.
Topics: Animals; Polyadenylation; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Cell Nucleus; RNA, Messenger; Mammals
PubMed: 36416579
DOI: 10.1002/2211-5463.13528 -
Molecular Cell Jun 2022Viegas et al. (2022) discover that in Trypanosoma brucei the poly(A) tails of the variant surface glycoprotein (VSG) transcripts are methylated, a mechanism that...
Viegas et al. (2022) discover that in Trypanosoma brucei the poly(A) tails of the variant surface glycoprotein (VSG) transcripts are methylated, a mechanism that stabilizes these transcripts and ensures protection against the immune response in mammals.
Topics: Animals; Mammals; Membrane Glycoproteins; Poly A; RNA, Messenger; Trypanosoma brucei brucei; Variant Surface Glycoproteins, Trypanosoma
PubMed: 35659324
DOI: 10.1016/j.molcel.2022.05.017 -
Developmental Cell Apr 2024During oocyte maturation and early embryogenesis, changes in mRNA poly(A)-tail lengths strongly influence translation, but how these tail-length changes are orchestrated...
During oocyte maturation and early embryogenesis, changes in mRNA poly(A)-tail lengths strongly influence translation, but how these tail-length changes are orchestrated has been unclear. Here, we performed tail-length and translational profiling of mRNA reporter libraries (each with millions of 3' UTR sequence variants) in frog oocytes and embryos and in fish embryos. Contrasting to previously proposed cytoplasmic polyadenylation elements (CPEs), we found that a shorter element, UUUUA, together with the polyadenylation signal (PAS), specify cytoplasmic polyadenylation, and we identified contextual features that modulate the activity of both elements. In maturing oocytes, this tail lengthening occurs against a backdrop of global deadenylation and the action of C-rich elements that specify tail-length-independent translational repression. In embryos, cytoplasmic polyadenylation becomes more permissive, and additional elements specify waves of stage-specific deadenylation. Together, these findings largely explain the complex tapestry of tail-length changes observed in early frog and fish development, with strong evidence of conservation in both mice and humans.
Topics: Animals; Oocytes; Polyadenylation; Protein Biosynthesis; Poly A; 3' Untranslated Regions; RNA, Messenger; Gene Expression Regulation, Developmental; Mice; Humans; Embryo, Nonmammalian; Embryonic Development; Female; Xenopus laevis; Cytoplasm
PubMed: 38460509
DOI: 10.1016/j.devcel.2024.02.007 -
Genes & Development Aug 2023The mRNA 3' poly(A) tail plays a critical role in regulating both mRNA translation and turnover. It is bound by the cytoplasmic poly(A) binding protein (PABPC), an...
The mRNA 3' poly(A) tail plays a critical role in regulating both mRNA translation and turnover. It is bound by the cytoplasmic poly(A) binding protein (PABPC), an evolutionarily conserved protein that can interact with translation factors and mRNA decay machineries to regulate gene expression. Mammalian PABPC1, the prototypical PABPC, is expressed in most tissues and interacts with eukaryotic translation initiation factor 4G (eIF4G) to stimulate translation in specific contexts. In this study, we uncovered a new mammalian PABPC, which we named neural PABP (neuPABP), as it is predominantly expressed in the brain. neuPABP maintains a unique architecture as compared with other PABPCs, containing only two RNA recognition motifs (RRMs) and maintaining a unique N-terminal domain of unknown function. neuPABP expression is activated in neurons as they mature during synaptogenesis, where neuPABP localizes to the soma and postsynaptic densities. neuPABP interacts with the noncoding RNA BC1, as well as mRNAs coding for ribosomal and mitochondrial proteins. However, in contrast to PABPC1, neuPABP does not associate with actively translating mRNAs in the brain. In keeping with this, we show that neuPABP has evolved such that it does not bind eIF4G and as a result fails to support protein synthesis in vitro. Taken together, these results indicate that mammals have expanded their PABPC repertoire in the brain and propose that neuPABP may support the translational repression of select mRNAs.
Topics: Animals; Eukaryotic Initiation Factor-4G; Poly(A)-Binding Proteins; Neurons; Brain; Mammals
PubMed: 37704377
DOI: 10.1101/gad.350597.123 -
BioEssays : News and Reviews in... Jun 2021Post-transcriptional regulation faces a distinctive challenge in gametes. Transcription is limited when the germ cells enter the division phase due to condensed... (Review)
Review
Post-transcriptional regulation faces a distinctive challenge in gametes. Transcription is limited when the germ cells enter the division phase due to condensed chromatin, while gene expression during gamete maturation, fertilization, and early cleavage depends on existing mRNA post-transcriptional coordination. The dynamics of the 3'-poly(A) tail play crucial roles in defining mRNA fate. The 3'-poly(A) tail is covered with poly(A)-binding proteins (PABPs) that help to mediate mRNA metabolism and recent work has shed light on the number and function of germ cell-specific expressed PABPs. There are two structurally different PABP groups distinguished by their cytoplasmic and nuclear localization. Both lack catalytic activity but are coupled with various roles through their interaction with multifunctional partners during mRNA metabolism. Here, we present a synopsis of PABP function during gametogenesis and early embryogenesis and describe both conventional and current models of the functions and regulation of PABPs, with an emphasis on the physiological significance of how germ cell-specific PABPs potentially affect human fertility.
Topics: Cell Nucleus; Embryonic Development; Gametogenesis; Humans; Poly(A)-Binding Proteins; RNA, Messenger
PubMed: 33830517
DOI: 10.1002/bies.202000335 -
Biochemical and Biophysical Research... Aug 2022Eukaryotic translation is a complex process that involves the interplay of various translation factors to convert genetic information into a specific amino acid chain....
Eukaryotic translation is a complex process that involves the interplay of various translation factors to convert genetic information into a specific amino acid chain. According to an elegant model of eukaryotic translation initiation, the 3' poly(A) tail of an mRNA, which is occupied by poly(A)-binding proteins (PABPs), communicates with the 5'-cap bound by eIF4E to enhance translation. Although the circularization of mRNA resulting from the communication is widely understood, it has yet to be directly observed. To explore mRNA circularization in translation, we analyzed the level of colocalization of eIF4E, eIF4G, and PABP on individual mRNAs in polysomal and subpolysomal fractions using single polysome analysis. Our results show that the three tested proteins barely coexist in mRNA in either polysomal or subpolysomal fractions, implying that the closed-loop structure generated by the communication between eIF4E, eIF4G, and PAPB may be transient during translation.
Topics: Eukaryotic Initiation Factor-4E; Eukaryotic Initiation Factor-4G; Poly(A)-Binding Proteins; Polyribosomes; Protein Binding; Protein Biosynthesis; RNA, Messenger; Ribonucleoproteins
PubMed: 35716598
DOI: 10.1016/j.bbrc.2022.06.017 -
RNA (New York, N.Y.) May 2022Poly(A) tail length is regulated in both the nucleus and cytoplasm. One factor that controls polyadenylation in the cytoplasm is CPEB1, an RNA binding protein that...
Poly(A) tail length is regulated in both the nucleus and cytoplasm. One factor that controls polyadenylation in the cytoplasm is CPEB1, an RNA binding protein that associates with specific mRNA 3'UTR sequences to tether enzymes that add and remove poly(A). Two of these enzymes, the noncanonical poly(A) polymerases GLD2 (TENT2, PAPD4, Wispy) and GLD4 (TENT4B, PAPD5, TRF4, TUT3), interact with CPEB1 to extend poly(A). To identify additional RNA binding proteins that might anchor GLD4 to RNA, we expressed double tagged GLD4 in U87MG cells, which was used for sequential immunoprecipitation and elution followed by mass spectrometry. We identified several RNA binding proteins that coprecipitated with GLD4, among which was FMRP. To assess whether FMRP regulates polyadenylation, we performed TAIL-seq from WT and FMRP-deficient HEK293 cells. Surprisingly, loss of FMRP resulted in an overall increase in poly(A), which was also observed for several specific mRNAs. Conversely, loss of CPEB1 elicited an expected decrease in poly(A), which was examined in cultured neurons. We also examined polyadenylation in wild type (WT) and FMRP-deficient mouse brain cortex by direct RNA nanopore sequencing, which identified RNAs with both increased and decreased poly(A). Our data show that FMRP has a role in mediating poly(A) tail length, which adds to its repertoire of RNA regulation.
Topics: Animals; HEK293 Cells; Humans; Mice; Poly A; Polyadenylation; Polynucleotide Adenylyltransferase; RNA, Messenger; RNA-Binding Proteins; Transcription Factors; mRNA Cleavage and Polyadenylation Factors
PubMed: 35217597
DOI: 10.1261/rna.079050.121 -
BMC Genomics Jul 2022Genome-wide RNA-sequencing technologies are increasingly critical to a wide variety of diagnostic and research applications. RNA-seq users often first enrich for mRNA,...
BACKGROUND
Genome-wide RNA-sequencing technologies are increasingly critical to a wide variety of diagnostic and research applications. RNA-seq users often first enrich for mRNA, with the most popular enrichment method being poly(A) selection. In many applications it is well-known that poly(A) selection biases the view of the transcriptome by selecting for longer tailed mRNA species.
RESULTS
Here, we show that poly(A) selection biases Oxford Nanopore direct RNA sequencing. As expected, poly(A) selection skews sequenced mRNAs toward longer poly(A) tail lengths. Interestingly, we identify a population of mRNAs (> 10% of genes' mRNAs) that are inconsistently captured by poly(A) selection due to highly variable poly(A) tails, and demonstrate this phenomenon in our hands and in published data. Importantly, we show poly(A) selection is dispensable for Oxford Nanopore's direct RNA-seq technique, and demonstrate successful library construction without poly(A) selection, with decreased input, and without loss of quality.
CONCLUSIONS
Our work expands the utility of direct RNA-seq by validating the use of total RNA as input, and demonstrates important technical artifacts from poly(A) selection that inconsistently skew mRNA expression and poly(A) tail length measurements.
Topics: High-Throughput Nucleotide Sequencing; Poly A; Polyadenylation; RNA; RNA, Messenger; Sequence Analysis, RNA; Transcriptome
PubMed: 35869428
DOI: 10.1186/s12864-022-08762-8 -
International Journal of Molecular... Sep 2022Poly(A) tails are present on almost all eukaryotic mRNAs, and play critical roles in mRNA stability, nuclear export, and translation efficiency. The biosynthesis and... (Review)
Review
Poly(A) tails are present on almost all eukaryotic mRNAs, and play critical roles in mRNA stability, nuclear export, and translation efficiency. The biosynthesis and shortening of a poly(A) tail are regulated by large multiprotein complexes. However, the molecular mechanisms of these protein machineries still remain unclear. Recent studies regarding the structural and biochemical characteristics of those protein complexes have shed light on the potential mechanisms of polyadenylation and deadenylation. This review summarizes the recent structural studies on pre-mRNA 3'-end processing complexes that initiate the polyadenylation and discusses the similarities and differences between yeast and human machineries. Specifically, we highlight recent biochemical efforts in the reconstitution of the active human canonical pre-mRNA 3'-end processing systems, as well as the roles of RBBP6/Mpe1 in activating the entire machinery. We also describe how poly(A) tails are removed by the PAN2-PAN3 and CCR4-NOT deadenylation complexes and discuss the emerging role of the cytoplasmic poly(A)-binding protein (PABPC) in promoting deadenylation. Together, these recent discoveries show that the dynamic features of these machineries play important roles in regulating polyadenylation and deadenylation.
Topics: DNA-Binding Proteins; Exoribonucleases; Humans; Multiprotein Complexes; Poly(A)-Binding Proteins; Polyadenylation; RNA Precursors; RNA Stability; RNA, Messenger; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Ubiquitin-Protein Ligases; mRNA Cleavage and Polyadenylation Factors
PubMed: 36232288
DOI: 10.3390/ijms231910985 -
Methods in Enzymology 2021Transcription of mRNAs culminates in RNA cleavage and a coordinated polyadenylation event at the 3' end. In its journey to be translated, the resulting transcript is...
Transcription of mRNAs culminates in RNA cleavage and a coordinated polyadenylation event at the 3' end. In its journey to be translated, the resulting transcript is under constant regulation by cap-binding proteins, miRNAs, and RNA binding proteins, including poly(A) binding proteins (PABPs). The interplay between all these factors determines whether nuclear or cytoplasmic exoribonucleases will gain access to and remove the poly(A) tail, which is so critical to the stability and translation capacity of the mRNA. In this chapter, we present an overview of two of the key features of the mRNA life-cycle: cleavage/polyadenylation and deadenylation, and describe biochemical assays that have been generated to study the activity of each of these enzymatic reactions. Finally, we also provide protocols to investigate mRNA's poly(A) length. The importance of these assays is highlighted by the dynamic and essential role the poly(A) tail length plays in controlling gene expression.
Topics: Exoribonucleases; Poly A; Polyadenylation; RNA, Messenger; RNA-Binding Proteins
PubMed: 34183126
DOI: 10.1016/bs.mie.2021.04.005