A Quantitative and Predictive Model for RNA Binding by Human Pumilio Proteins

SummaryHigh-throughput methodologies have enabled routine generation of RNA target sets and sequence motifs for RNA-binding proteins (RBPs). Nevertheless, quantitative approaches are needed to capture the landscape of RNA/RBP interactions responsible for cellular regulation. We have used the RNA-MaP platform to directly measure equilibrium binding for thousands of designed RNAs and to construct a predictive model for RNA recognition by the human Pumilio proteins PUM1 and PUM2. Despite prior findings of linear sequence motifs, our measurements revealed widespread residue flipping and instances of positional coupling. Application of our thermodynamic model to published in vivo crosslinking data reveals quantitative agreement between predicted affinities and in vivo occupancies. Our analyses suggest a thermodynamically driven, continuous Pumilio binding landscape that is negligibly affected by RNA structure or kinetic factors, such as displacement by ribosomes. This work provides a quantitative foundation for dissecting the cellular behavior of RBPs and cellular features that impact their occupancies.

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Human Pumilio proteins directly bind the CCR4-NOT deadenylase complex to regulate the transcriptome

10.1101/2020.11.17.387456 ◽

2020 ◽

Author(s):

Isioma I.I. Enwerem ◽

Nathan D. Elrod ◽

Chung-Te Chang ◽

Ai Lin ◽

Ping Ji ◽

...

Keyword(s):

Rna Binding ◽

Rna Binding Proteins ◽

Molecular Model ◽

Direct Interaction ◽

Vertebrate Development ◽

Regulate Gene Expression ◽

Mrna Decapping ◽

Specific Mrnas ◽

Decapping Enzyme ◽

Pumilio Proteins

AbstractPumilio paralogs, PUM1 and PUM2, are sequence-specific RNA-binding proteins that are essential for vertebrate development and neurological functions. PUM1&2 negatively regulate gene expression by accelerating degradation of specific mRNAs. Here, we determined the repression mechanism and impact of human PUM1&2 on the transcriptome. We identified subunits of the CCR4-NOT (CNOT) deadenylase complex required for stable interaction with PUM1&2 and to elicit CNOT-dependent repression. Isoform-level RNA sequencing revealed broad co-regulation of target mRNAs through the PUM-CNOT repression mechanism.Functional dissection of the domains of PUM1&2 identified a conserved N-terminal region that confers the predominant repressive activity via direct interaction with CNOT. In addition, we show that the mRNA decapping enzyme, DCP2, has an important role in repression by PUM1&2 N-terminal regions. Our results support a molecular model of repression by human PUM1&2 via direct recruitment of CNOT deadenylation machinery in a decapping-dependent mRNA decay pathway.

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Structural features within the NORAD long noncoding RNA underlie efficient repression of Pumilio activity

10.1101/2021.11.19.469243 ◽

2021 ◽

Author(s):

Omer Ziv ◽

Svetlana Farberov ◽

Jian You Lau ◽

Eric A Miska ◽

Grzegorz Kudla ◽

...

Keyword(s):

Rna Structure ◽

Mammalian Cells ◽

Noncoding Rna ◽

Rna Binding ◽

Rna Binding Proteins ◽

Structural Features ◽

Structural Domain ◽

Mrna Targets ◽

Structural Principles ◽

Pumilio Proteins

It is increasingly appreciated that long non-coding RNAs (lncRNAs) carry out important functions in mammalian cells, but how these are encoded in their sequences and manifested in their structures remains largely unknown. Some lncRNAs bind to and modulate the availability of RNA binding proteins, but the structural principles that underlie this mode of regulation are underexplored. Here, we focused on the NORAD lncRNA, which binds Pumilio proteins and modulates their ability to repress hundreds of mRNA targets. We probed the RNA structure and long-range RNA-RNA interactions formed by NORAD inside cells, under different stressful conditions. We discovered that NORAD structure is highly modular, and consists of well-defined domains that contribute independently to NORAD function. We discovered that NORAD structure spatially clusters the Pumilio binding sites along NORAD in a manner that contributes to the de-repression of Pumilio target proteins. Following arsenite stress, the majority of NORAD structure undergoes relaxation and forms inter-molecular interactions with RNAs that are targeted to stress granules. NORAD sequence thus dictates elaborated structural domain organization that facilitates its function on multiple levels, and which helps explain the extensive evolutionary sequence conservation of NORAD regions that are not predicted to directly bind Pumilio proteins.

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A conserved abundant cytoplasmic long noncoding RNA modulates repression by Pumilio proteins in human cells

10.1101/033423 ◽

2015 ◽

Cited By ~ 1

Author(s):

Ailone Tichon ◽

Noa Gil ◽

Yoav Lubelsky ◽

Tal Havkin Solomon ◽

Doron Lemze ◽

...

Keyword(s):

Cell Division ◽

Human Genome ◽

Chromosome Segregation ◽

Binding Sites ◽

Long Noncoding Rna ◽

Noncoding Rna ◽

Rna Binding ◽

Rna Binding Proteins ◽

Mrna Levels ◽

Pumilio Proteins

AbstractThousands of long noncoding RNA (lncRNA) genes are encoded in the human genome, and hundreds of them are evolutionary conserved, but their functions and modes of action remain largely obscure. Particularly enigmatic lncRNAs are those that are exported to the cytoplasm, including NORAD – an abundant and highly conserved cytoplasmic lncRNA. Most of the sequence of NORAD is comprised of repetitive units that together contain at least 17 functional binding sites for the two Pumilio homologs in mammals. Through binding to PUM1 and PUM2, NORAD modulates the mRNA levels of their targets, which are enriched for genes involved in chromosome segregation during cell division. Our results suggest that some cytoplasmic lncRNAs function by modulating the activities of RNA binding proteins, an activity which positions them at key junctions of cellular signaling pathways.

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PUMILIO hyperactivity drives premature aging of Norad-deficient mice

eLife ◽

10.7554/elife.42650 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 18

Author(s):

Florian Kopp ◽

Mahmoud M Elguindy ◽

Mehmet E Yalvac ◽

He Zhang ◽

Beibei Chen ◽

...

Keyword(s):

Rna Binding ◽

Rna Binding Proteins ◽

Noncoding Rnas ◽

Rapid Onset ◽

Premature Aging ◽

Transcriptional Repressors ◽

Mouse Tissues ◽

Physiologic Function ◽

Pumilio Proteins

Although numerous long noncoding RNAs (lncRNAs) have been identified, our understanding of their roles in mammalian physiology remains limited. Here, we investigated the physiologic function of the conserved lncRNA Norad in vivo. Deletion of Norad in mice results in genomic instability and mitochondrial dysfunction, leading to a dramatic multi-system degenerative phenotype resembling premature aging. Loss of tissue homeostasis in Norad-deficient animals is attributable to augmented activity of PUMILIO proteins, which act as post-transcriptional repressors of target mRNAs to which they bind. Norad is the preferred RNA target of PUMILIO2 (PUM2) in mouse tissues and, upon loss of Norad, PUM2 hyperactively represses key genes required for mitosis and mitochondrial function. Accordingly, enforced Pum2 expression fully phenocopies Norad deletion, resulting in rapid-onset aging-associated phenotypes. These findings provide new insights and open new lines of investigation into the roles of noncoding RNAs and RNA binding proteins in normal physiology and aging.

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Structural and functional similarities and differences in nucleolar Pumilio RNA-binding proteins between Arabidopsis and Charophyte Chara corallina

10.21203/rs.2.17798/v1 ◽

2019 ◽

Author(s):

Su Hyun Park ◽

Hyung-Sae im ◽

Prakash Jyoti Kalita ◽

Sang-Bong Choi

Keyword(s):

Binding Proteins ◽

Rna Binding ◽

Tertiary Structure ◽

Rna Binding Proteins ◽

Chara Corallina ◽

Its2 Sequence ◽

Random Coil ◽

Rrna Processing ◽

Morphological Defects ◽

Pumilio Proteins

Abstract Background: Pumilio RNA-binding proteins are evolutionarily conserved throughout all eukaryotes and are involved in RNA decay, transport, and translation repression in the cytoplasm. Even though a majority of Pumilio proteins function in the cytoplasm, two nucleolar forms have been reported to have a function in rRNA processing in Arabidopsis. Chara is known to be most closely related to land plants as it shares several characteristics with modern embryophytes. Results: In this study, we identified two putative nucleolar Pumilio protein genes, ChPUM2 and ChPUM3 , from the transcriptome of Chara corallina . Of the two ChPUM proteins, ChPUM2 was the most similar to Arabidopsis APUM23 in the amino acid sequence (27% identity and 45% homology) and the predicted protein structure, while ChPUM3 was similar to APUM24 (35% identity and 54% homology). The transient expression of 35S:ChPUM2-RFP and 35S:ChPUM3-RFP showed nucleolar localization of fusion proteins in tobacco leaf cells, similar to the expression of APUM23-GFP and APUM24-GFP. Moreover, 35S:ChPUM2 complemented the morphological defects of the apum23 phenotypes but not those of apum24, while 35S:ChPUM3 could not complement the apum23 and apum24 mutants. Similarly, the 35S:ChPUM2/apum23 plants rescued the pre-rRNA processing defect of apum23 , but 35S:ChPUM3/apum24 +/- plants did not rescue that of apum24 . Consistent with these complementation results, a known target RNA-binding sequence at the end of the 18S rRNA (5'-GAAUUGACGG) for APUM23 was conserved in Arabidopsis and Chara, whereas a target region of ITS2 pre-rRNA for APUM24 was 156 nt longer in Chara than in Arabidopsis. Moreover, ChPUM2 and APUM23 were predicted to have nearly identical structures, but ChPUM3 and APUM24 have different structures in the 5th C-terminal Puf RNA-binding domain as ChPUM3 has a long random coil in this domain. Conclusions: Our results indicate that ChPUM2 has evolutionarily maintained functions in Arabidopsis, while ChPUM3 is not functional in Arabidopsis, most likely due to the long target ITS2 sequence of Arabidopsis and the distinct tertiary structure from Arabidopsis homologue APUM24.

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Structural and functional similarities and differences in nucleolar Pumilio RNA-binding proteins between Arabidopsis and the charophyte Chara corallina

10.21203/rs.2.17798/v3 ◽

2020 ◽

Author(s):

Su Hyun Park ◽

Hyung-Sae Kim ◽

Prakash Jyoti Kalita ◽

Sang-Bong Choi

Keyword(s):

Protein Structure ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Chara Corallina ◽

Random Coil ◽

Rrna Processing ◽

High Coverage ◽

Morphological Defects ◽

Pumilio Proteins

Abstract Background: Pumilio RNA-binding proteins are evolutionarily conserved throughout eukaryotes and are involved in RNA decay, transport, and translation repression in the cytoplasm. Although a majority of Pumilio proteins function in the cytoplasm, two nucleolar forms have been reported to have a function in rRNA processing in Arabidopsis. The species of the genus Chara have been known to be most closely related to land plants, as they share several characteristics with modern Embryophyta. Results: In this study, we identified two putative nucleolar Pumilio protein genes, namely, ChPUM2 and ChPUM3, from the transcriptome of Chara corallina. Of the two ChPUM proteins, ChPUM2 was most similar in amino acid sequence (27% identity and 45% homology) and predicted protein structure to Arabidopsis APUM23, while ChPUM3 was similar to APUM24 (35% identity and 54% homology). The transient expression of 35S:ChPUM2-RFP and 35S:ChPUM3-RFP showed nucleolar localization of fusion proteins in tobacco leaf cells, similar to the expression of 35S:APUM23-GFP and 35S:APUM24-GFP. Moreover, 35S:ChPUM2 complemented the morphological defects of the apum23 phenotypes but not those of apum24, while 35S:ChPUM3 could not complement the apum23 and apum24 mutants. Similarly, the 35S:ChPUM2/apum23 plants rescued the pre-rRNA processing defect of apum23, but 35S:ChPUM3/apum24+/- plants did not rescue that of apum24. Consistent with these complementation results, a known target RNA-binding sequence at the end of the 18S rRNA (5'-GGAAUUGACGG) for APUM23 was conserved in Arabidopsis and C. corallina, whereas a target region of ITS2 pre-rRNA for APUM24 was 156 nt longer in C. corallina than in A. thaliana. Moreover, ChPUM2 and APUM23 were predicted to have nearly identical structures, but ChPUM3 and APUM24 have different structures in the 5th C-terminal Puf RNA-binding domain, which had a longer random coil in ChPUM3 than in APUM24. Conclusions: ChPUM2 of C. corallina was functional in Arabidopsis, similar to APUM23, but ChPUM3 did not substitute for APUM24 in Arabidopsis. Protein homology modeling showed high coverage between APUM23 and ChPUM2, but displayed structural differences between APUM24 and ChPUM3. Together with the protein structure of ChPUM3 itself, a short ITS2 of Arabidopsis pre-rRNA may interrupt the binding of ChPUM3 to 3’-extended 5.8S pre-rRNA.

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PUMILIO hyperactivity drives premature aging of Norad-deficient mice

10.1101/432112 ◽

2018 ◽

Author(s):

Florian Kopp ◽

Mehmet E. Yalvac ◽

Beibei Chen ◽

He Zhang ◽

Sungyul Lee ◽

...

Keyword(s):

Rna Binding ◽

Rna Binding Proteins ◽

Noncoding Rnas ◽

Rapid Onset ◽

Premature Aging ◽

Transcriptional Repressors ◽

Mouse Tissues ◽

Physiologic Function ◽

Pumilio Proteins

ABSTRACTAlthough numerous long noncoding RNAs (lncRNAs) have been identified, our understanding of their roles in mammalian physiology remains limited. Here we investigated the physiologic function of the conserved lncRNA Norad in vivo. Deletion of Norad in mice results in genomic instability and mitochondrial dysfunction, leading to a dramatic multi-system degenerative phenotype resembling premature aging. Loss of tissue homeostasis in Norad-deficient animals is attributable to augmented activity of PUMILIO proteins, which act as post-transcriptional repressors of target mRNAs to which they bind. Norad is the preferred RNA target of PUMILIO2 (PUM2) in mouse tissues and, upon loss of Norad, PUM2 hyperactively represses key genes required for mitosis and mitochondrial function. Remarkably, enforced Pum2 expression fully phenocopies Norad deletion, resulting in rapid-onset aging-associated phenotypes. These findings provide new insights and open new lines of investigation into the roles of noncoding RNAs and RNA binding proteins in normal physiology and aging.

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Structural and functional similarities and differences in nucleolar Pumilio RNA-binding proteins between Arabidopsis and the charophyte Chara corallina

10.21203/rs.2.17798/v2 ◽

2020 ◽

Author(s):

Su Hyun Park ◽

Hyung-Sae Kim ◽

Prakash Jyoti Kalita ◽

Sang-Bong Choi

Keyword(s):

Protein Structure ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Chara Corallina ◽

Random Coil ◽

Rrna Processing ◽

High Coverage ◽

Morphological Defects ◽

Pumilio Proteins

Abstract Background: Pumilio RNA-binding proteins are evolutionarily conserved throughout eukaryotes and are involved in RNA decay, transport, and translation repression in the cytoplasm. Although a majority of Pumilio proteins function in the cytoplasm, two nucleolar forms have been reported to have a function in rRNA processing in Arabidopsis. The species of the genus Chara have been known to be most closely related to land plants, as they share several characteristics with modern Embryophyta.Results: In this study, we identified two putative nucleolar Pumilio protein genes, namely, ChPUM2 and ChPUM3, from the transcriptome of Chara corallina. Of the two ChPUM proteins, ChPUM2 was most similar in amino acid sequence (27% identity and 45% homology) and predicted protein structure to Arabidopsis APUM23, while ChPUM3 was similar to APUM24 (35% identity and 54% homology). The transient expression of 35S:ChPUM2-RFP and 35S:ChPUM3-RFP showed nucleolar localization of fusion proteins in tobacco leaf cells, similar to the expression of 35S:APUM23-GFP and 35S:APUM24-GFP. Moreover, 35S:ChPUM2 complemented the morphological defects of the apum23 phenotypes but not those of apum24, while 35S:ChPUM3 could not complement the apum23 and apum24 mutants. Similarly, the 35S:ChPUM2/apum23 plants rescued the pre-rRNA processing defect of apum23, but 35S:ChPUM3/apum24+/- plants did not rescue that of apum24. Consistent with these complementation results, a known target RNA-binding sequence at the end of the 18S rRNA (5'-GGAAUUGACGG) for APUM23 was conserved in Arabidopsis and C. corallina, whereas a target region of ITS2 pre-rRNA for APUM24 was 156 nt longer in C. corallina than in A. thaliana. Moreover, ChPUM2 and APUM23 were predicted to have nearly identical structures, but ChPUM3 and APUM24 have different structures in the 5th C-terminal Puf RNA-binding domain, which had a longer random coil in ChPUM3 than in APUM24.Conclusions: ChPUM2 of C. corallina was functional in Arabidopsis, similar to APUM23, but ChPUM3 did not substitute for APUM24 in Arabidopsis. Protein homology modeling showed high coverage between APUM23 and ChPUM2, but displayed structural differences between APUM24 and ChPUM3. Together with the protein structure of ChPUM3 itself, a short ITS2 of Arabidopsis pre-rRNA may interrupt the binding of ChPUM3 to 3’-extended 5.8S pre-rRNA.

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