Structure of the Rna15 RRM–RNA complex reveals the molecular basis of GU specificity in transcriptional 3′-end processing factors

Pancevac, C.; Goldstone, David C.; Ramos, Andrés; Taylor, Ian A.

doi:10.1093/nar/gkq002

Cited by 52 publications

(91 citation statements)

References 32 publications

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“…This structure completed earlier studies of Hrp1 with the enhancer EE element (15) and Rna15 with GU-rich RNA sequences (22) and of CstF-64 (23). To better understand how 3′-end processing signals are recognized to initiate 3′-end processing and transcription termination, we sought to obtain structural information on the complete CF I complex including Rna14, Rna15, Hrp1, and the pre-mRNA transcript.…”

Section: Resultssupporting

confidence: 67%

Structural and biochemical analysis of the assembly and function of the yeast pre-mRNA 3′ end processing complex CF I

Barnwal

Lee

Moore

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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The accuracy of the 3′-end processing by cleavage and polyadenylation is essential for mRNA biogenesis and transcription termination. In yeast, two poorly conserved neighboring elements upstream of cleavage sites are important for accuracy and efficiency of this process. These two RNA sequences are recognized by the RNA binding proteins Hrp1 and Rna15, but efficient processing in vivo requires a bridging protein (Rna14), which forms a stable dimer of heterodimers with Rna15 to stabilize the RNA-protein complex. We earlier reported the structure of the ternary complex of Rna15 and Hrp1 bound to the RNA processing element. We now report the use of solution NMR to study the interaction of Hrp1 with the Rna14-Rna15 heterodimer in the presence and absence of 3′-end processing signals. By using methyl selective labeling on Hrp1, in vivo activity and pull-down assays, we were able to study this complex of several hundred kDa, identify the interface within Hrp1 responsible for recruitment of Rna14 and validate the functional significance of this interaction through structure-driven mutational analysis.mRNA processing | mRNA transport | protein-RNA | methyl-TROSY | modeling E ukaryotic messenger RNA precursors (pre-mRNA) undergo extensive cotranscriptional processing before their transport to the cytoplasm and subsequent translation (1, 2). The processing events include 5′-end capping, splicing to remove intronic sequences and cleavage and polyadenylation at the 3′-end (3, 4). Correct 3′-end processing is necessary for mRNA biogenesis and transcription termination, preserves RNAs from degradation (5), promotes its export to the cytoplasm (6), enhances translation (7) and promotes transcriptional activation (4, 8). Cleavage and polyadenylation can be uncoupled in vitro but in vivo they are connected with each other and with transcription termination. Defects in 3′-end processing have also been associated with several diseases in humans (9), but this process may be even more critical in yeast that has shorter intergenic regions and much rarer alternative splicing (10, 11). In this organism, efficient mRNA transport is mostly dependent on polyadenylation rather than splicing.In yeast, the 3′-end processing reaction is executed by a complex machine containing more than 20 proteins (4) which share significant similarities with human 3′-end processing proteins. The entire yeast 3′-end processing complex is divided into two subcomplexes, called cleavage factor I (CF I) and cleavage and polyadenylation factor (CPF) (4). CF I binds to the pre-mRNA upstream of the cleavage signal within the pre-mRNA, whereas CPF binds at the cleavage site as well as upstream and downstream of the cleavage site. Several sequences within yeast pre-mRNAs, which are surprisingly poorly conserved, direct the recruitment of cleavage and polyadenylation factors (12, 13). In the 5′-3′ direction, they include AU-rich efficiency element (EE) responsible for polyadenylation efficiency; A-rich positioning element (PE) critical for precise 3′-end processing; t...

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Section: Resultssupporting

confidence: 67%

Structural and biochemical analysis of the assembly and function of the yeast pre-mRNA 3′ end processing complex CF I

Barnwal

Lee

Moore

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…For further details, see Materials and Methods. motif (RRM) domain that binds GU-rich RNA (41,63). Hrp1 has two RRMs that bind to AU-containing RNAs (67).…”

Section: Discussionmentioning

confidence: 99%

“…Since deletion of the Spt5 CTR led to a marked reduction in the occupancy of CFI subunits, but not to a complete loss, we asked which factors contribute to the residual binding of CFI. Since Rna15 and Hrp1 contain RNA recognition motifs that are known to bind RNA sequences in vitro (33,41,63,67), we reasoned that nascent RNA may contribute to CFI recruitment. To address this, we performed an RNase-ChIP assay (1).…”

Section: Investigation Of Elongation Factor Recruitment By Spt5 Ctrmentioning

confidence: 99%

The Spt5 C-Terminal Region Recruits Yeast 3′ RNA Cleavage Factor I

Mayer

Schreieck

Lidschreiber

et al. 2012

Molecular and Cellular Biology

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During transcription of protein-coding genes, RNA polymerase II (Pol II) associates with many factors, including elongation factors, RNA-processing factors, and chromatin-modifying enzymes (42,66,80). Many of these factors are recruited by binding to the C-terminal repeat domain (CTD), a tail-like extension of the largest Pol II subunit that is highly phosphorylated during elongation (10,18,54). Some elongation factors, including TFIIS (34) and Spt5 (36, 51), also bind the body of Pol II.The gene encoding Spt5 was identified in Saccharomyces cerevisiae as a suppressor of transposon insertion in the promoter region of the HIS4 gene (86). Spt5 is an essential nuclear protein (77) and binds Spt4 (24). Spt5 associates with Pol II in vivo, and mutations lead to a slow-growth phenotype in the presence of the nucleotidedepleting drug 6-azauracil (6-AU), arguing for a role for Spt5 in transcription elongation (24). The human homolog of yeast Spt4/5 affects Pol II elongation (83). Chromatin immunoprecipitation (ChIP) revealed that Spt5 colocalizes with Pol II throughout the transcribed region and past the polyadenylation (pA) site (35,52,68,81). Spt4/5 is present on all transcribed yeast genes and is a general component of the elongation complex (52, 81). Spt4/5 also associates with Pol I (82) and Pol I genes (74).Spt5 is the only known Pol II-associated factor that is conserved in all three kingdoms of life (85). The bacterial Spt5 homolog NusG and archaeal Spt5 consist of an N-terminal (NGN) domain and a flexibly linked C-terminal Kyrpides-Ouzounis-Woese (KOW) domain (38, 59). The structures of archaeal Spt4/5 bound to RNA polymerase or its clamp domain are known (36, 51). These structures indicate that the NGN domain closes the active center cleft to lock nucleic acids and render the elongation complex stable and processive (26,36,51). Eukaryotic Spt5 possesses additional regions and domains. Yeast Spt5 consists of an acidic N-terminal region, followed by an NGN domain, five KOW domains, and a repetitive C-terminal region (CTR) (69,77,89).Spt5 has recently emerged as a platform that recruits factors to elongating Pol II. Spt5 copurifies with over 90 yeast proteins that are involved in transcription elongation, RNA processing, transcription termination, and mRNA export (44). Spt4/5 interacts with the histone chaperone Spt6 to modulate chromatin structure (24,78) Deletion of the Spt5 CTR in yeast is not lethal (16,45,89) but leads to sensitivity to 6-AU and a slow-growth phenotype at 16°C (45, 89). The CTR deletion is synthetically lethal with the deletion of the gene for the Pol II CTD kinase Ctk1 (45). Deletion of the CTR in fission yeast leads to a slow-growth phenotype and abnormal cell morphology (75). The slow-growth phenotype is intensified if the Pol II CTD is truncated (75), suggesting that the CTR cooperates with the CTD. Deletion of the CTR impairs embryo-

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“…The Rna15 RRM preferentially binds to a U-rich or GUrich sequence in vitro (95,97). The affinity for the RNA is generally low (92) but can be significantly enhanced by Rna14 or Hrp1 (a yeast CFI component that binds the EE, no mammalian homolog) (92,98).…”

Section: Cstfmentioning

confidence: 99%

Delineating the Structural Blueprint of the Pre-mRNA 3′-End Processing Machinery

Xiang

Tong

Manley

2014

Molecular and Cellular Biology

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Processing of mRNA precursors (pre-mRNAs) by polyadenylation is an essential step in gene expression. Polyadenylation consists of two steps, cleavage and poly(A) synthesis, and requires multiple cis elements in the pre-mRNA and a megadalton protein complex bearing the two essential enzymatic activities. While genetic and biochemical studies remain the major approaches in characterizing these factors, structural biology has emerged during the past decade to help understand the molecular assembly and mechanistic details of the process. With structural information about more proteins and higher-order complexes becoming available, we are coming closer to obtaining a structural blueprint of the polyadenylation machinery that explains both how this complex functions and how it is regulated and connected to other cellular processes.

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Structure of the Rna15 RRM–RNA complex reveals the molecular basis of GU specificity in transcriptional 3′-end processing factors

Cited by 52 publications

References 32 publications

Structural and biochemical analysis of the assembly and function of the yeast pre-mRNA 3′ end processing complex CF I

Structural and biochemical analysis of the assembly and function of the yeast pre-mRNA 3′ end processing complex CF I

The Spt5 C-Terminal Region Recruits Yeast 3′ RNA Cleavage Factor I

Delineating the Structural Blueprint of the Pre-mRNA 3′-End Processing Machinery

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