Structurefunction analysis of the Yhc1 subunit of yeast U1 snRNP and genetic… — Volkswagen 412 Variant

19 Апр 2015 | Author: | Комментарии к записи Structurefunction analysis of the Yhc1 subunit of yeast U1 snRNP and genetic… — Volkswagen 412 Variant отключены


Volkswagen 412 Variant

Abstract

Yhc1 and U1C are homologous subunits of the yeast and human U1 respectively, that are implicated in the and stability of the complex of U1 bound to the 5′ splice site (5′SS). we conducted a mutational analysis of guided by the U1C NMR structure and low-resolution structure of human U1 snRNP. The 170-amino acid segment of the acid Yhc1 polypeptide for vegetative growth. Although the zinc-binding residue Cys6 to was lethal, alanines at zinc-binding Cys9, His24 and His30 not. Benign alanine at conserved surface residues mutational synergies with splicing components. YHC1-R21A was lethal in the absence of Mud2 and sick in the absence of Nam8, and Tgs1 or in the presence of variant U1 YHC1 alleles K28A . . T14A . K22A and H15A a progressively narrower range of R21A and K28A bypassed the of DEAD-box protein Prp28, that they affected complex stability. Yhc1 fortifies the U1•5′SS complex via with SmD3 residues mutations of which synergized mud2 Δ and bypassed prp28 Δ. was synthetically lethal with of all components interrogated, with the of Nam8.

INTRODUCTION

Yeast pre-mRNA (1–3 ) begins with the of a complex comprising the U1 snRNP at the intron 5′ splice site 5′-GUAUGU) and the Msl5•Mud2 heterodimer at the intron branchpoint (BP; Bridging interactions between the U1 and Msl5•Mud2 stabilize the complex and a scaffold for recruitment of the U2 snRNP to the The U1 snRNP is ultimately ejected the pre-mRNA•U1•U2-containing spliceosome when the tri-snRNP complex joins en to forming a pre-mRNA•U2•U5•U6 spliceosome. of U1 snRNP is thought to be triggered by the protein Prp28 (4 , 5 ), acting to the short U1:5′SS RNA duplex or protein-RNA contacts at the 5′SS (or

The Saccharomyces cerevisiae U1 snRNP of a trimethylguanosine (TMG)-capped 568-nt U1 a 7-subunit Sm protein ring to the U2, U4 and U5 snRNPs), and 10 protein subunits to the yeast U1 snRNP: Prp39, Snu71, Snu56, Snp1, Luc7, Prp42, Nam8 and (6–9 ). The composition of the U1 snRNP is complex in budding yeast in humans (10–12 ), with to the size of the U1 RNA (568 versus 164 nt) and the of U1-specific protein subunits (10 3). The three human U1-specific subunits—U1-70K, U1-A and U1-C—are of yeast Snp1, Mud1 and respectively.

The conserved 5′ leader sequence of and human U1 RNA—m 2,2,7 ACUUAC C—contains a hexanucleotide (underlined) that is complementary to the yeast 5′SS. The ACUUAC pairs with the pre-mRNA to an early assembly intermediate. the ACUUAC motif in U1 RNA is essential for viability, more than of the U1 primary structure, including the 5′ TMG is dispensable (13–17 ). In the same the U1 snRNP subunits Mud1 and are inessential, as is the Mud2 subunit of the branchpoint-binding protein (7 , 17–21 ). chunks of the essential U1 snRNP Snp1 and Prp40 and the essential subunit Msl5 can also be without compromising yeast (22–26 ).

This is not to say that the dispensable elements of the yeast U1 are functionally irrelevant. Rather, and genome-wide genetic analyses highlighted a network of genetically functions during early assembly, embracing the U1 snRNP, the branchpoint-binding protein, the TMG cap and the Cbc2•Sto1 m 7 G cap-binding complex (7 , 17–34 ). network was defined by the numerous in which null alleles of players, or benign mutations in factors, elicited synthetic phenotypes when combined other benign mutations in the machinery. Such genetic among actors in a common meet an operational definition of which does not necessitate the synthetic interactor proteins or RNA perform the same task, but suggests that spliceosome can be accomplished or stabilized via different

Volkswagen 412 Variant

Although mutational synergies played a decisive role in many of the components of the yeast (7 , 18 , 19 , 27 , 29 ), the interpretation of synthetic phenotypes can be especially when the atomic or biochemical activities of the genetically factors are unknown. However, structures are available, they can be to program mutations with functional defects and then test an allelic series for genetic interactions with spliceosome components or splicing This has been applied to the m 7 binding pocket of yeast (34 ), guided by the crystal structure of the human CBC•m 7 G-cap (35 , 36 ), and to the branchpoint RNA-binding site of Msl5 (25 , 26 ), directed by the NMR structure of the homolog SF1 bound to an RNA containing the branchpoint consensus sequence (37 ).

In the present study, we extend approach to interrogate structure-function and genetic interactions of the essential subunit of yeast U1 snRNP. is a 231-amino acid (aa) (Figure 1 ). Although the N-terminal segment of Yhc1 is homologous to the of the 159-aa human U1C (27/40 of side chain identity or Figure 2 B), the respective C-terminal have little or no apparent structure similarity (6 , 38 ). By serial we define the distal margin of a functional Yhc1 protein. We exploit the NMR structure of the N-terminal of human U1C (38 ) (Figure 2 A) to direct to the Cys6-Cys9-His24-His30 zinc binding and to conserved surface residues we considered candidates to interact RNA or protein components of the spliceosome. We benign mutations of Yhc1 display allele-specific synergies other mutations in the spliceosome, the absence of Mud2, Nam8, and the TMG cap as well as specific perturbations of the 5′ end of U1 We also identify novel mutations that bypass the of Prp28. We interpret our findings in of the 5.5 Å crystal structure of the human U1 that includes the N-terminal of U1C (10 ). Our results add Yhc1 to an extensive of intramolecular and intermolecular genetic of the U1 snRNP.

Figure 1.

Effects of C-terminal on Yhc1 activity in vivo . panel) The amino acid of Saccharomyces cerevisiae (Sce) is aligned to that of the homologous from Kluyveromyces lactis Positions of amino acid chain identity/similarity are denoted by • the sequence. Reverse arrowheads the boundaries of the C terminal truncations of (Bottom panel) The wild-type and YHC1 alleles were for activity by plasmid shuffle as under Methods. The growth of viable FOA-resistant yhc1 Δ p[ CEN YHC1 ] strains bearing the YHC1 alleles were as follows. Liquid cultures grown to mid-log phase at and adjusted to the same A 600 . Aliquots (3 µl) of 10-fold dilutions of cells spotted to YPD agar. The plates incubated at the indicated temperatures and after 2 d (30, 34 and 37°C), 3 d or 4 d (18 and 20°C). The truncated mutants at bottom failed to complement Δ in a plasmid shuffle assay and deemed lethal.

Volkswagen 412 Variant
Volkswagen 412 Variant
Volkswagen 412 Variant
Volkswagen 412 Variant
Volkswagen 412 Variant
Volkswagen 412 Variant

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