Insight into Mammalian Selenocysteine Insertion: Domain Structure and Ribosome Binding Properties of Sec Insertion Sequence Binding Protein 2

ABSTRACT The cotranslational incorporation of the unusual amino acid selenocysteine (Sec) into both prokaryotic and eukaryotic proteins requires the recoding of a UGA stop codon as one specific for Sec. The recognition of UGA as Sec in mammalian selenoproteins requires a Sec insertion sequence (SECIS) element in the 3′ untranslated region as well as the SECIS binding protein SBP2. Here we report a detailed analysis of SBP2 structure and function using truncation and site-directed mutagenesis. We have localized the RNA binding domain to a conserved region shared with several ribosomal proteins and eukaryotic translation termination release factor 1. We also identified a separate and novel functional domain N-terminal to the RNA binding domain which was required for Sec insertion but not for SECIS binding. Conversely, we showed that the RNA binding domain was necessary but not sufficient for Sec insertion and that the conserved glycine residue within this domain was required for SECIS binding. Using glycerol gradient sedimentation, we found that SBP2 was stably associated with the ribosomal fraction of cell lysates and that this interaction was not dependent on its SECIS binding activity. This interaction also occurred with purified components in vitro, and we present data which suggest that the SBP2-ribosome interaction occurs via 28S rRNA. SBP2 may, therefore, have a distinct function in selecting the ribosomes to be used for Sec insertion.

Download Full-text

RNA-binding domain of the A protein component of the U1 small nuclear ribonucleoprotein analyzed by NMR spectroscopy is structurally similar to ribosomal proteins.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.88.6.2495 ◽

1991 ◽

Vol 88 (6) ◽

pp. 2495-2499 ◽

Cited By ~ 83

Author(s):

D. W. Hoffman ◽

C. C. Query ◽

B. L. Golden ◽

S. W. White ◽

J. D. Keene

Keyword(s):

Nmr Spectroscopy ◽

Ribosomal Proteins ◽

Rna Binding ◽

Binding Domain ◽

Protein Component ◽

Small Nuclear Ribonucleoprotein ◽

Rna Binding Domain ◽

Nuclear Ribonucleoprotein

Download Full-text

Site-directed mutagenesis of the double-stranded RNA binding domain of bacterially-expressed σ3 reovirus protein

Virus Research ◽

10.1016/0168-1702(96)01281-6 ◽

1996 ◽

Vol 41 (2) ◽

pp. 141-151 ◽

Cited By ~ 17

Author(s):

Q Wang ◽

J Bergeron ◽

T Mabrouk ◽

G Lemay

Keyword(s):

Rna Binding ◽

Binding Domain ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Double Stranded Rna ◽

Rna Binding Domain

Download Full-text

3′ End Processing of Drosophilamelanogaster Histone Pre-mRNAs: Requirement for Phosphorylated Drosophila Stem-Loop Binding Protein and Coevolution of the Histone Pre-mRNA Processing System

Molecular and Cellular Biology ◽

10.1128/mcb.22.18.6648-6660.2002 ◽

2002 ◽

Vol 22 (18) ◽

pp. 6648-6660 ◽

Cited By ~ 33

Author(s):

Zbigniew Dominski ◽

Xiao-cui Yang ◽

Christy S. Raska ◽

Carlos Santiago ◽

Christoph H. Borchers ◽

...

Keyword(s):

Insect Cells ◽

Rna Binding ◽

Binding Protein ◽

Mrna Processing ◽

Binding Domain ◽

Loop Structure ◽

Stem Loop ◽

Stem Loop Structure ◽

Nuclear Extracts ◽

Rna Binding Domain

ABSTRACT Synthetic pre-mRNAs containing the processing signals encoded by Drosophila melanogaster histone genes undergo efficient and faithful endonucleolytic cleavage in nuclear extracts prepared from Drosophila cultured cells and 0- to 13-h-old embryos. Biochemical requirements for the in vitro cleavage are similar to those previously described for the 3′ end processing of mammalian histone pre-mRNAs. Drosophila 3′ end processing does not require ATP and occurs in the presence of EDTA. However, in contrast to mammalian processing, Drosophila processing generates the final product ending four nucleotides after the stem-loop. Cleavage of the Drosophila substrates is abolished by depleting the extract of the Drosophila stem-loop binding protein (dSLBP), indicating that both dSLBP and the stem-loop structure in histone pre-mRNA are essential components of the processing machinery. Recombinant dSLBP expressed in insect cells by using the baculovirus system efficiently complements the depleted extract. Only the RNA-binding domain plus the 17 amino acids at the C terminus of dSLBP are required for processing. The full-length dSLBP expressed in insect cells is quantitatively phosphorylated on four residues in the C-terminal region. Dephosphorylation of the recombinant dSLBP reduces processing activity. Human and Drosophila SLBPs are not interchangeable and strongly inhibit processing in the heterologous extracts. The RNA-binding domain of the dSLBP does not substitute for the RNA-binding domain of the human SLBP in histone pre-mRNA processing in mammalian extracts. In addition to the stem-loop structure and dSLBP, 3′ processing in Drosophila nuclear extracts depends on the presence of a short stretch of purines located ca. 20 nucleotides downstream from the stem, and an Sm-reactive factor, most likely the Drosophila counterpart of vertebrate U7 snRNP.

Download Full-text

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.

Download Full-text