U7 deciphered: the mechanism that forms the unusual 3′ end of metazoan replication-dependent histone mRNAs

Biochemical Society Transactions ◽

10.1042/bst20210323 ◽

2021 ◽

Author(s):

Zbigniew Dominski ◽

Liang Tong

Keyword(s):

Animal Cells ◽

Cleavage Reaction ◽

Stem Loop ◽

Histone Mrnas ◽

Stem Loop Structure ◽

U7 Snrnp ◽

Eukaryotic Mrnas ◽

Cleavage And Polyadenylation ◽

Processing Mechanisms ◽

Processing Mechanism

In animal cells, replication-dependent histone mRNAs end with a highly conserved stem–loop structure followed by a 4- to 5-nucleotide single-stranded tail. This unique 3′ end distinguishes replication-dependent histone mRNAs from all other eukaryotic mRNAs, which end with a poly(A) tail produced by the canonical 3′-end processing mechanism of cleavage and polyadenylation. The pioneering studies of Max Birnstiel's group demonstrated nearly 40 years ago that the unique 3′ end of animal replication-dependent histone mRNAs is generated by a distinct processing mechanism, whereby histone mRNA precursors are cleaved downstream of the stem–loop, but this cleavage is not followed by polyadenylation. The key role is played by the U7 snRNP, a complex of a ∼60 nucleotide U7 snRNA and many proteins. Some of these proteins, including the enzymatic component CPSF73, are shared with the canonical cleavage and polyadenylation machinery, justifying the view that the two metazoan pre-mRNA 3′-end processing mechanisms have a common evolutionary origin. The studies on U7 snRNP culminated in the recent breakthrough of reconstituting an entirely recombinant human machinery that is capable of accurately cleaving histone pre-mRNAs, and determining its structure in complex with a pre-mRNA substrate (with 13 proteins and two RNAs) that is poised for the cleavage reaction. The structure uncovered an unanticipated network of interactions within the U7 snRNP and a remarkable mechanism of activating catalytically dormant CPSF73 for the cleavage. This work provides a conceptual framework for understanding other eukaryotic 3′-end processing machineries.

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ZFP100, a Component of the Active U7 snRNP Limiting for Histone Pre-mRNA Processing, Is Required for Entry into S Phase

Molecular and Cellular Biology ◽

10.1128/mcb.00391-06 ◽

2006 ◽

Vol 26 (17) ◽

pp. 6702-6712 ◽

Cited By ~ 22

Author(s):

Eric J. Wagner ◽

William F. Marzluff

Keyword(s):

Fluorescent Protein ◽

S Phase ◽

Mrna Processing ◽

Limiting Factor ◽

Stem Loop ◽

Histone Mrnas ◽

U7 Snrnp ◽

Eukaryotic Mrnas ◽

Green Fluorescent

ABSTRACT Metazoan replication-dependent histone mRNAs are the only eukaryotic mRNAs that are not polyadenylated. The cleavage of histone pre-mRNA to form the unique 3′ end requires the U7 snRNP and the stem-loop binding protein (SLBP) that binds the 3′ end of histone mRNA. U7 snRNP contains three novel proteins, Lsm10 and Lsm11, which are part of the core U7 Sm complex, and ZFP100, a Zn finger protein that helps stabilize binding of the U7 snRNP to the histone pre-mRNA by interacting with the SLBP/pre-mRNA complex. Using a reporter gene that encodes a green fluorescent protein mRNA ending in a histone 3′ end and mimics histone gene expression, we demonstrate that ZFP100 is the limiting factor for histone pre-mRNA processing in vivo. The overexpression of Lsm10 and Lsm11 increases the cellular levels of U7 snRNP but has no effect on histone pre-mRNA processing, while increasing the amount of ZFP100 increases histone pre-mRNA processing but has no effect on U7 snRNP levels. We also show that knocking down the known components of U7 snRNP by RNA interference results in a reduction in cell growth and an unsuspected cell cycle arrest in early G1, suggesting that active U7 snRNP is necessary to allow progression through G1 phase to S phase.

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Cotranscriptional Processing of Drosophila Histone mRNAs

Molecular and Cellular Biology ◽

10.1128/mcb.23.12.4046-4055.2003 ◽

2003 ◽

Vol 23 (12) ◽

pp. 4046-4055 ◽

Cited By ~ 19

Author(s):

Todd E. Adamson ◽

David H. Price

Keyword(s):

Transcription Initiation ◽

Divalent Cations ◽

Histone H4 ◽

Stem Loop ◽

Histone Mrnas ◽

Stem Loop Structure ◽

U7 Snrnp ◽

Processing Site ◽

The One

ABSTRACT The 3′ ends of metazoan histone mRNAs are generated by specialized processing machinery that cleaves downstream of a conserved stem-loop structure. To examine how this reaction might be influenced by transcription, we used a Drosophila melanogaster in vitro system that supports both processes. In this system the complete synthesis of histone mRNA, including transcription initiation and elongation, followed by 3′ end formation, occurred at a physiologically significant rate. Processing of free transcripts was efficient and occurred with a t 1/2 of less than 1 min. Divalent cations were not required, but nucleoside triphosphates (NTPs) stimulated the rate of cleavage slightly. Isolated elongation complexes encountered a strong arrest site downstream of the mature histone H4 3′ end. In the presence of NTPs, transcripts in these arrested complexes were processed at a rate similar to that of free RNA. Removal of NTPs dramatically reduced this rate, potentially due to concealment of the U7 snRNP binding element. The arrest site was found to be a conserved feature located 32 to 35 nucleotides downstream of the processing site on the H4, H2b, and H3 genes. The significance of the newly discovered arrest sites to our understanding of the coupling between transcription and RNA processing on the one hand and histone gene expression on the other is discussed.

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Conservation of the pentanucleotide motif at the top of the yellow fever virus 17D 3′ stem–loop structure is not required for replication

Journal of General Virology ◽

10.1099/vir.0.82811-0 ◽

2007 ◽

Vol 88 (6) ◽

pp. 1738-1747 ◽

Cited By ~ 19

Author(s):

Patrícia A. G. C. Silva ◽

Richard Molenkamp ◽

Tim J. Dalebout ◽

Nathalie Charlier ◽

Johan H. Neyts ◽

...

Keyword(s):

Yellow Fever ◽

Molecular Mechanisms ◽

Yellow Fever Virus ◽

Animal Cell ◽

Fever Virus ◽

Loop Structure ◽

Animal Cells ◽

Pn Sequence ◽

Stem Loop ◽

Stem Loop Structure

The pentanucleotide (PN) sequence 5′-CACAG-3′ at the top of the 3′ stem–loop structure of the flavivirus genome is well conserved in the arthropod-borne viruses but is more variable in flaviviruses with no known vector. In this study, the sequence requirements of the PN motif for yellow fever virus 17D (YFV) replication were determined. In general, individual mutations at either the second, third or fourth positions were tolerated and resulted in replication-competent virus. Mutations at the fifth position were lethal. Base pairing of the nucleotide at the first position of the PN motif and a nucleotide four positions downstream of the PN (ninth position) was a major determinant for replication. Despite the fact that the majority of the PN mutants were able to replicate efficiently, they were outcompeted by parental YFV-17D virus following repeated passages in double-infected cell cultures. Surprisingly, some of the virus mutants at the first and/or the ninth position that maintained the possibility of forming a base pair were found to have a similar fitness to YFV-17D under these conditions. Overall, these experiments suggest that YFV is less dependent on sequence conservation of the PN motif for replication in animal cells than West Nile virus. However, in animal cell culture, YFV has a preference for the wt CACAG PN sequence. The molecular mechanisms behind this preference remain to be elucidated.

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Two Xenopus Proteins That Bind the 3′ End of Histone mRNA: Implications for Translational Control of Histone Synthesis during Oogenesis

Molecular and Cellular Biology ◽

10.1128/mcb.19.1.835 ◽

1999 ◽

Vol 19 (1) ◽

pp. 835-845 ◽

Cited By ~ 52

Author(s):

Zeng-Feng Wang ◽

Thomas C. Ingledue ◽

Zbigniew Dominski ◽

Ricardo Sanchez ◽

William F. Marzluff

Keyword(s):

Translational Control ◽

Mrna Processing ◽

Loop Structure ◽

Histone Mrna ◽

Stem Loop ◽

Histone Mrnas ◽

Stem Loop Structure ◽

Histone Synthesis

ABSTRACT Translationally inactive histone mRNA is stored in frog oocytes, and translation is activated at oocyte maturation. The replication-dependent histone mRNAs are not polyadenylated and end in a conserved stem-loop structure. There are two proteins (SLBPs) which bind the 3′ end of histone mRNA in frog oocytes. SLBP1 participates in pre-mRNA processing in the nucleus. SLBP2 is oocyte specific, is present in the cytoplasm, and does not support pre-mRNA processing in vivo or in vitro. The stored histone mRNA is bound to SLBP2. As oocytes mature, SLBP2 is degraded and a larger fraction of the histone mRNA is bound to SLBP1. The mechanism of activation of translation of histone mRNAs may involve exchange of SLBPs associated with the 3′ end of histone mRNA.

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The Histone 3′-Terminal Stem-Loop-Binding Protein Enhances Translation through a Functional and Physical Interaction with Eukaryotic Initiation Factor 4G (eIF4G) and eIF3

Molecular and Cellular Biology ◽

10.1128/mcb.22.22.7853-7867.2002 ◽

2002 ◽

Vol 22 (22) ◽

pp. 7853-7867 ◽

Cited By ~ 48

Author(s):

Jun Ling ◽

Simon J. Morley ◽

Virginia M. Pain ◽

William F. Marzluff ◽

Daniel R. Gallie

Keyword(s):

Binding Protein ◽

Initiation Factor ◽

Translational Efficiency ◽

Physical Interaction ◽

Loop Structure ◽

Eukaryotic Initiation Factor ◽

Stem Loop ◽

Histone Mrnas ◽

Cell Extracts ◽

Stem Loop Structure

ABSTRACT Metazoan cell cycle-regulated histone mRNAs are unique cellular mRNAs in that they terminate in a highly conserved stem-loop structure instead of a poly(A) tail. Not only is the stem-loop structure necessary for 3′-end formation but it regulates the stability and translational efficiency of histone mRNAs. The histone stem-loop structure is recognized by the stem-loop-binding protein (SLBP), which is required for the regulation of mRNA processing and turnover. In this study, we show that SLBP is required for the translation of mRNAs containing the histone stem-loop structure. Moreover, we show that the translation of mRNAs ending in the histone stem-loop is stimulated in Saccharomyces cerevisiae cells expressing mammalian SLBP. The translational function of SLBP genetically required eukaryotic initiation factor 4E (eIF4E), eIF4G, and eIF3, and expressed SLBP coisolated with S. cerevisiae initiation factor complexes that bound the 5′ cap in a manner dependent on eIF4G and eIF3. Furthermore, eIF4G coimmunoprecipitated with endogenous SLBP in mammalian cell extracts and recombinant SLBP and eIF4G coisolated. These data indicate that SLBP stimulates the translation of histone mRNAs through a functional interaction with both the mRNA stem-loop and the 5′ cap that is mediated by eIF4G and eIF3.

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Three Proteins of the U7-Specific Sm Ring Function as the Molecular Ruler To Determine the Site of 3′-End Processing in Mammalian Histone Pre-mRNA

Molecular and Cellular Biology ◽

10.1128/mcb.00296-09 ◽

2009 ◽

Vol 29 (15) ◽

pp. 4045-4056 ◽

Cited By ~ 16

Author(s):

Xiao-cui Yang ◽

Matthew P. Torres ◽

William F. Marzluff ◽

Zbigniew Dominski

Keyword(s):

Cleavage Site ◽

Dependent Manner ◽

Loop Structure ◽

Affinity Tag ◽

Stem Loop ◽

Stem Loop Structure ◽

5.8S Rrna ◽

Molecular Ruler ◽

U7 Snrnp ◽

And Function

ABSTRACT Cleavage of histone pre-mRNAs at the 3′ end is guided by the U7 snRNP, which is a component of a larger 3′-end processing complex. To identify other components of this complex, we isolated proteins that stably associate with a fragment of histone pre-mRNA containing all necessary processing elements and a biotin affinity tag at the 5′ end. Among the isolated proteins, we identified three well-characterized processing factors: the stem-loop binding protein (SLBP), which interacts with the stem-loop structure upstream of the cleavage site, and both Lsm11 and SmB, which are components of the U7-specifc Sm ring. We also identified 3′hExo/Eri-1, a multifunctional 3′ exonuclease that is known to trim the 3′ end of 5.8S rRNA. 3′hExo primarily binds to the downstream portion of the stem-loop structure in mature histone mRNA, with the upstream portion being occupied by SLBP. The two proteins bind their respective RNA sites in a cooperative manner, and 3′hExo can recruit SLBP to a mutant stem-loop that itself does not interact with SLBP. UV-cross-linking studies used to characterize interactions within the processing complex demonstrated that 3′hExo also interacts in a U7-dependent manner with unprocessed histone pre-mRNA. However, this interaction is not required for the cleavage reaction. The region between the cleavage site and the U7-binding site interacts with three low-molecular-weight proteins, which were identified as components of the U7-specific Sm core: SmB, SmD3, and Lsm10. These proteins likely rigidify the substrate and function as the molecular ruler in determining the site of cleavage.

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Mutations in the RNA Binding Domain of Stem-Loop Binding Protein Define Separable Requirements for RNA Binding and for Histone Pre-mRNA Processing

Molecular and Cellular Biology ◽

10.1128/mcb.21.6.2008-2017.2001 ◽

2001 ◽

Vol 21 (6) ◽

pp. 2008-2017 ◽

Cited By ~ 27

Author(s):

Zbigniew Dominski ◽

Judith A. Erkmann ◽

John A. Greenland ◽

William F. Marzluff

Keyword(s):

Rna Binding ◽

Binding Protein ◽

Binding Activity ◽

Mrna Processing ◽

Binding Domain ◽

Stem Loop ◽

Amino Acid Region ◽

Stem Loop Structure ◽

U7 Snrnp ◽

Rna Binding Domain

ABSTRACT Expression of replication-dependent histone genes at the posttranscriptional level is controlled by stem-loop binding protein (SLBP). One function of SLBP is to bind the stem-loop structure in the 3′ untranslated region of histone pre-mRNAs and facilitate 3′ end processing. Interaction of SLBP with the stem-loop is mediated by the centrally located RNA binding domain (RBD). Here we identify several highly conserved amino acids in the RBD mutation of which results in complete or substantial loss of SLBP binding activity. We also identify residues in the RBD which do not contribute to binding to the stem-loop RNA but instead are required for efficient recruitment of U7 snRNP to histone pre-mRNA. Recruitment of the U7 snRNP to the pre-mRNA also depends on the 20-amino-acid region located immediately downstream of the RBD. A critical region of the RBD contains the sequence YDRY. The tyrosines are required for RNA binding, and the DR dipeptide is essential for processing but not for RNA binding. It is likely that the RBD of SLBP interacts directly with both the stem-loop RNA and other processing factor(s), most likely the U7 snRNP, to facilitate histone pre-mRNA processing.

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Identification of the Major Spliceosomal RNAs in Dictyostelium discoideum Reveals Developmentally Regulated U2 Variants and Polyadenylated snRNAs

Eukaryotic Cell ◽

10.1128/ec.00065-06 ◽

2006 ◽

Vol 5 (6) ◽

pp. 924-934 ◽

Cited By ~ 27

Author(s):

Andrea Hinas ◽

Pontus Larsson ◽

Lotta Avesson ◽

Leif A. Kirsebom ◽

Anders Virtanen ◽

...

Keyword(s):

Dictyostelium Discoideum ◽

Noncoding Rna ◽

Gene Promoter ◽

Small Nuclear Rna ◽

Developmentally Regulated ◽

Stem Loop ◽

Stem Loop Structure ◽

Spliceosomal Rnas ◽

Nuclear Rna ◽

Eukaryotic Mrnas

ABSTRACT Most eukaryotic mRNAs depend upon precise removal of introns by the spliceosome, a complex of RNAs and proteins. Splicing of pre-mRNA is known to take place in Dictyostelium discoideum, and we previously isolated the U2 spliceosomal RNA experimentally. In this study, we identified the remaining major spliceosomal RNAs in Dictyostelium by a bioinformatical approach. Expression was verified from 17 small nuclear RNA (snRNA) genes. All these genes are preceded by a putative noncoding RNA gene promoter. Immunoprecipitation showed that snRNAs U1, U2, U4, and U5, but not U6, carry the conserved trimethylated 5′ cap structure. A number of divergent U2 species are expressed in Dictyostelium. These RNAs carry the U2 RNA hallmark sequence and structure motifs but have an additional predicted stem-loop structure at the 5′ end. Surprisingly, and in contrast to the other spliceosomal RNAs in this study, the new U2 variants were enriched in the cytoplasm and were developmentally regulated. Furthermore, all of the snRNAs could also be detected as polyadenylated species, and polyadenylated U1 RNA was demonstrated to be located in the cytoplasm.

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