Specificities of Caenorhabditis elegans and human hairpin binding proteins for the first nucleotide in the histone mRNA hairpin loop

Michel, Fabrice; Schümperli, Daniel; Müller, Berndt

doi:10.1017/s135583820000056x

Cited by 20 publications

(19 citation statements)

References 31 publications

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“…4C) together with the 3Ј-untranslated region of SLBP mRNA. This portion of SLBP contains the complete RNA-binding domain (residues 126-198), which alone is sufficient to bind the histone mRNA stem loop with high affinity (K d ϭ 4 nM) (35,36). PCR and sequence analyses of the cDNA library used for these experiments showed that it contained no other in-frame SLBP clone encoding the RNA-binding domain.…”

Section: Resultsmentioning

confidence: 99%

T7 phage display: A novel genetic selection system for cloning RNA-binding proteins from cDNA libraries

Danner

Belasco

2001

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

138

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RNA-binding proteins are central to posttranscriptional gene regulation and play an important role in a number of major human diseases. Cloning such proteins is a crucial but often difficult step in elucidating the biological function of RNA regulatory elements. To make it easier to clone proteins that specifically bind RNA elements of interest, we have developed a rapid and broadly applicable in vitro genetic selection method based on T7 phage display. Using hairpin II of U1 small nuclear RNA (U1hpII) or the 3 stem loop of histone mRNA as bait, we could selectively amplify T7 phage that display either the spliceosomal protein U1A or the histone stem loop-binding protein from a lung cDNA phage library containing more than 10 7 independent clones. The use of U1hpII mutants with various affinities for U1A revealed that this method allows the selection even of proteins that bind their cognate RNA targets with relatively weak affinities (Kd as high as the micromolar range). Experiments with a mixture of recombinant phage displaying U1A or the closely related protein U2B؆ demonstrated that addition of a competitor RNA can suppress selection of a protein with a higher affinity for a given RNA target, thereby allowing the preferential amplification of a lower affinity protein. Together, these findings suggest that T7 phage display can be used to rapidly and selectively clone virtually any protein that binds a known RNA regulatory element, including those that bind with low affinity or that must compete for binding with other proteins. R NA-binding proteins (RNA-BPs) play a key role in a variety of posttranscriptional regulatory processes, including RNA processing, nucleocytoplasmic transport, translation, and mRNA decay (1-3). Moreover, a burgeoning body of evidence has implicated a number of RNA-BPs in the genetic etiology of human diseases such as fragile X mental retardation (4), paraneoplastic neurologic disorders (5), and spinal muscular atrophy (6), as well as in many microbial infections, including AIDS (7) and influenza (8).An essential step in elucidating the regulatory mechanism of a given RNA element is to identify and characterize the protein(s) that binds to this element and mediates its effect. Characterization of such an RNA-BP is most readily accomplished by cloning its cDNA. Traditionally, this has been achieved by protein purification, peptide microsequencing, and cDNA amplification with PCR primers designed on the basis of the amino acid sequence information. This labor-intensive strategy requires an enormous effort and can be particularly difficult in the case of RNA-BPs present at a low cellular concentration.Some genetic screening methods have been developed to facilitate the cloning of RNA-BP cDNAs, most notably the yeast three-hybrid system (9, 10) and plaque-lift analysis (11,12). Both techniques have allowed the cloning of previously unidentified RNA-BPs (13-16). However, several limitations may interfere with the general applicability of these procedures. For example, when using the three-hyb...

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Section: Resultsmentioning

confidence: 99%

T7 phage display: A novel genetic selection system for cloning RNA-binding proteins from cDNA libraries

Danner

Belasco

2001

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

138

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“…The 15 amino acids in the N-terminal region of xSLBP1 required for translation are identical in xSLBP1 and the mammalian SLBPs, consistent with this activation being physiologically important in mammalian cells. There is not a similar sequence in the Drosophila (55) or C. elegans SLBP (33,37), and the Drosophila SLBP is not active in translation of the Luc-SL mRNA in the reticulocyte lysate (unpublished results).…”

Section: Discussionmentioning

confidence: 97%

The Stem-Loop Binding Protein Is Required for Efficient Translation of Histone mRNA In Vivo and In Vitro

Sánchez

Marzluff

2002

Molecular and Cellular Biology

120

186

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Metazoan replication-dependent histone mRNAs end in a conserved stem-loop rather than in the poly(A) tail found on all other mRNAs. The 3 end of histone mRNA binds a single class of proteins, the stem-loop binding proteins (SLBP). In Xenopus, there are two SLBPs: xSLBP1, the homologue of the mammalian SLBP, which is required for processing of histone pre-mRNA, and xSLBP2, which is expressed only during oogenesis and is bound to the stored histone mRNA in Xenopus oocytes. The stem-loop is required for efficient translation of histone mRNAs and substitutes for the poly(A) tail, which is required for efficient translation of other eucaryotic mRNAs. When a rabbit reticulocyte lysate is programmed with uncapped luciferase mRNA ending in the histone stem-loop, there is a three-to sixfold increase in translation in the presence of xSLBP1 while xSLBP2 has no effect on translation. Neither SLBP affected the translation of a luciferase mRNA ending in a mutant stem-loop that does not bind SLBP. Capped luciferase mRNAs ending in the stem-loop were injected into Xenopus oocytes after overexpression of either xSLBP1 or xSLBP2. Overexpression of xSLBP1 in the oocytes stimulated translation, while overexpression of xSLBP2 reduced translation of the luciferase mRNA ending in the histone stem-loop. A small region in the N-terminal portion of xSLBP1 is required to stimulate translation both in vivo and in vitro. An MS2-human SLBP1 fusion protein can activate translation of a reporter mRNA ending in an MS2 binding site, indicating that xSLBP1 only needs to be recruited to the 3 end of the mRNA but does not need to be directly bound to the histone stem-loop to activate translation.Replication-dependent histone mRNAs are unique among metazoan mRNAs because they are not polyadenylated but end, instead, in a highly conserved stem-loop (35). In contrast, histone mRNAs from fungi and plants are polyadenylated. Histone pre-mRNA processing requires only a single endonucleolytic cleavage to form the 3Ј end of the mature mRNA immediately after a highly conserved stem-loop structure (16). Most of the regulation of histone mRNA levels is mediated by the stem-loop and occurs at the posttranscriptional level, via regulation of mRNA processing and stability (22,44). It is likely that the stem-loop also fulfills the essential functions that are performed by the poly(A) tail on other mRNAs. One of these functions is to enhance the efficiency of translation (27,52). For example, the stem-loop has been shown to promote localization of histone mRNAs to polyribosomes (56). mRNAs ending in the histone stem-loop at the 3Ј end are translationally more active than messages ending in other stem-loops when transfected into Chinese hamster ovary cells (15).By using the yeast three-hybrid system, proteins that bind to the stem-loop have been isolated from several species (34,55,64,66). Although only a single SLBP is present in mammals (34, 66), Drosophila (55), and Caenorhabditis elegans, frog oocytes (46) express two SLBPs, called xSLBP1 and xSLBP2 (64). The ...

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“…Homologs of SLBP have been identified in human, mouse, frog, fruit fly, and Caenorhabditis elegans (Wang et al 1996;Michel et al 2000;Sullivan et al 2001). These SLBP homologs are known to contain a functionally important and centrally located RNA-binding domain, i.e., a domain interacting with the stem-loop structure.…”

Section: Homologs To the Metazoan Slbp Protein Are Identified In Protmentioning

confidence: 99%

Early evolution of histone mRNA 3′ end processing

López¹,

Samuelsson²

2007

RNA

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The replication-dependent histone mRNAs in metazoa are not polyadenylated, in contrast to the bulk of mRNA. Instead, they contain an RNA stem-loop (SL) structure close to the 39 end of the mature RNA, and this 39 end is generated by cleavage using a machinery involving the U7 snRNP and protein factors such as the stem-loop binding protein (SLBP). This machinery of 39 end processing is related to that of polyadenylation as protein components are shared between the systems. It is commonly believed that histone 39 end processing is restricted to metazoa and green algae. In contrast, polyadenylation is ubiquitous in Eukarya. However, using computational approaches, we have now identified components of histone 39 end processing in a number of protozoa. Thus, the histone mRNA stem-loop structure as well as the SLBP protein are present in many different protozoa, including Dictyostelium, alveolates, Trypanosoma, and Trichomonas. These results show that the histone 39 end processing machinery is more ancient than previously anticipated and can be traced to the root of the eukaryotic phylogenetic tree. We also identified histone mRNAs from both metazoa and protozoa that are polyadenylated but also contain the signals characteristic of histone 39 end processing. These results provide further evidence that some histone genes are regulated at the level of 39 end processing to produce either polyadenylated RNAs or RNAs with the 39 end characteristic of replicationdependent histone mRNAs.

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Specificities of Caenorhabditis elegans and human hairpin binding proteins for the first nucleotide in the histone mRNA hairpin loop

Cited by 20 publications

References 31 publications

T7 phage display: A novel genetic selection system for cloning RNA-binding proteins from cDNA libraries

T7 phage display: A novel genetic selection system for cloning RNA-binding proteins from cDNA libraries

The Stem-Loop Binding Protein Is Required for Efficient Translation of Histone mRNA In Vivo and In Vitro

Early evolution of histone mRNA 3′ end processing

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