RESUMEN
Human Cleavage Factor Im (CFI(m)) is an essential component of the pre-mRNA 3' processing complex that functions in the regulation of poly(A) site selection through the recognition of UGUA sequences upstream of the poly(A) site. Although the highly conserved 25 kDa subunit (CFI(m)25) of the CFI(m) complex possesses a characteristic alpha/beta/alpha Nudix fold, CFI(m)25 has no detectable hydrolase activity. Here we report the crystal structures of the human CFI(m)25 homodimer in complex with UGUAAA and UUGUAU RNA sequences. CFI(m)25 is the first Nudix protein to be reported to bind RNA in a sequence-specific manner. The UGUA sequence contributes to binding specificity through an intramolecular G:A Watson-Crick/sugar-edge base interaction, an unusual pairing previously found to be involved in the binding specificity of the SAM-III riboswitch. The structures, together with mutational data, suggest a novel mechanism for the simultaneous sequence-specific recognition of two UGUA elements within the pre-mRNA. Furthermore, the mutually exclusive binding of RNA and the signaling molecule Ap(4)A (diadenosine tetraphosphate) by CFI(m)25 suggests a potential role for small molecules in the regulation of mRNA 3' processing.
Asunto(s)
ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Escisión y Poliadenilación de ARNm/química , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secuencia de Bases , Sitios de Unión/genética , Cristalografía por Rayos X , Humanos , Enlace de Hidrógeno , Técnicas In Vitro , Cinética , Sustancias Macromoleculares/química , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Filogenia , Multimerización de Proteína , Procesamiento Postranscripcional del ARN , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Electricidad Estática , Factores de Escisión y Poliadenilación de ARNm/genéticaRESUMEN
3'-end cleavage and subsequent polyadenylation are critical steps in mRNA maturation. The precise location where cleavage occurs (referred to as poly(A) site) is determined by a tripartite mechanism in which a A(A/U)UAAA hexamer, GU rich downstream element and UGUA upstream element are recognized by the cleavage and polyadenylation factor (CPSF), cleavage stimulation factor (CstF) and cleavage factor I(m) (CFI(m)), respectively. CFI(m) is composed of a smaller 25 kDa subunit (CFI(m)25) and a larger 59, 68 or 72 kDa subunit. CFI(m)68 interacts with CFI(m)25 through its N-terminal RNA recognition motif (RRM). We recently solved the crystal structures of CFI(m)25 bound to RNA and of a complex of CFI(m)25, the RRM domain of CFI(m)68 and RNA. Our study illustrated the molecular basis for UGUA recognition by the CFI(m) complex, suggested a possible mechanism for CFI(m) mediated alternative polyadenylation, and revealed potential links between CFI(m) and other mRNA processing factors, such as the 20 kDa subunit of the cap binding protein (CBP20), and the splicing regulator U2AF65.
Asunto(s)
Factor de Estimulación del Desdoblamiento/metabolismo , Caperuzas de ARN , Empalme del ARN , ARN Mensajero/metabolismo , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Sitios de Unión/genética , Factor de Estimulación del Desdoblamiento/química , Factor de Estimulación del Desdoblamiento/genética , Humanos , Proteínas Nucleares/metabolismo , Poliadenilación , Estructura Secundaria de Proteína , Caperuzas de ARN/genética , Procesamiento Postranscripcional del ARN , Sitios de Empalme de ARN/genética , Ribonucleoproteínas/metabolismo , Factor de Empalme U2AF , Factores de Escisión y Poliadenilación de ARNm/química , Factores de Escisión y Poliadenilación de ARNm/genéticaRESUMEN
Cleavage factor I(m) (CFI(m)) is a highly conserved component of the eukaryotic mRNA 3' processing machinery that functions in sequence-specific poly(A) site recognition through the collaboration of a 25 kDa subunit containing a Nudix domain and a larger subunit of 59, 68, or 72 kDa containing an RNA recognition motif (RRM). Our previous work demonstrated that CFI(m)25 is both necessary and sufficient for sequence-specific binding of the poly(A) site upstream element UGUA. Here, we report the crystal structure of CFI(m)25 complexed with the RRM domain of CFI(m)68 and RNA. The CFI(m)25 dimer is clasped on opposite sides by two CFI(m)68 RRM domains. Each CFI(m)25 subunit binds one UGUA element specifically. Biochemical analysis indicates that the CFI(m)68 RRMs serve to enhance RNA binding and facilitate RNA looping. The intrinsic ability of CFI(m) to direct RNA looping may provide a mechanism for its function in the regulation of alternative poly(A) site selection.
Asunto(s)
Poli A/metabolismo , Subunidades de Proteína/metabolismo , ARN Mensajero/metabolismo , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Regiones no Traducidas 3' , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión/genética , Cristalización , Cristalografía por Rayos X , Enzimas Reparadoras del ADN/química , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Monoéster Fosfórico Hidrolasas/química , Unión Proteica/genética , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Relación Estructura-Actividad Cuantitativa , ARN Mensajero/química , Factores de Escisión y Poliadenilación de ARNm/química , Factores de Escisión y Poliadenilación de ARNm/genéticaRESUMEN
At least half of all human pre-mRNAs are subject to alternative 3' processing that may modulate both the coding capacity of the message and the array of post-transcriptional regulatory elements embedded within the 3' UTR. Vertebrate poly(A) site selection appears to rely primarily on the binding of CPSF to an A(A/U)UAAA hexamer upstream of the cleavage site and CstF to a downstream GU-rich element. At least one-quarter of all human poly(A) sites, however, lack the A(A/U)UAAA motif. We report that sequence-specific RNA binding of the human 3' processing factor CFI(m) can function as a primary determinant of poly(A) site recognition in the absence of the A(A/U)UAAA motif. CFI(m) is sufficient to direct sequence-specific, A(A/U)UAAA-independent poly(A) addition in vitro through the recruitment of the CPSF subunit hFip1 and poly(A) polymerase to the RNA substrate. ChIP analysis indicates that CFI(m) is recruited to the transcription unit, along with CPSF and CstF, during the initial stages of transcription, supporting a direct role for CFI(m) in poly(A) site recognition. The recognition of three distinct sequence elements by CFI(m), CPSF, and CstF suggests that vertebrate poly(A) site definition is mechanistically more similar to that of yeast and plants than anticipated.
Asunto(s)
Poli A/metabolismo , Regiones no Traducidas 3' , Animales , Secuencia de Bases , Sitios de Unión , Humanos , Inmunoprecipitación , Datos de Secuencia Molecular , Polinucleotido Adenililtransferasa/metabolismo , Homología de Secuencia de Ácido Nucleico , Transcripción Genética , VertebradosRESUMEN
Human cleavage factor I(m) (CFI(m)) is a heterodimeric RNA binding protein complex that functions at an early step in the assembly of the pre-mRNA 3' processing complex. In this report we show that CFI(m) can stimulate both cleavage and poly(A) addition, and can act to suppress poly(A) site cleavage in a sequence-dependent manner. Elevated levels of CFI(m) suppressed cleavage at the primary poly(A) site of the pre-mRNA encoding the 68 kDa subunit of CFI(m). CFI(m)-mediated suppression of poly(A) site cleavage was dependent upon the presence of three copies of an RNA element initially identified by CFI(m)-SELEX. These data provide evidence for a mechanism for the regulation of poly(A) site selection by a basal pre-mRNA 3' processing factor.