1H, 13C, and 15N resonance assignments for the N-terminal domain of Drosophila
Stem-Loop Binding Protein

Regulation of histone mRNA gene expression

The N-terminal domain of the Drosophila histone mRNA binding protein, SLBP, is intrinsically disordered with nascent helical structure.

Functional RNAs often form compact structures characterized by closely packed
helices. Crystallographic analysis of several large RNAs revealed a prevalent
interaction in which unpaired adenosine residues dock into the minor groove of
a receptor helix. This A-minor motif, potentially the most important element
responsible for global RNA architecture, has also been suggested to contribute
to the fidelity of protein synthesis by discriminating against near-cognate
tRNAs on the ribosome. The specificity of A-minor interactions is fundamental
to RNA tertiary structure formation, as well as to their proposed role in
translational accuracy. To investigate A-minor motif specificity, we analyzed
mutations in an A-minor interaction within the Tetrahymena group I
self-splicing intron. Thermodynamic and x-ray crystallographic results show
that the A-minor interaction strongly prefers canonical base pairs over base
mismatches in the receptor helix, enabling RNA interhelical packing through
specific recognition of Watson-Crick minor groove geometry.

Battle DJ, Doudna JA.
Specificity of RNA-RNA helix recognition.
Proc Natl Acad Sci U S A. 2002 Sep 3;99(18):11676-81. Epub 2002 Aug 20.

Proceedings of the National Academy of Sciences of the United States of America
Specificity of RNA-RNA helix recognition.

All metazoan messenger RNAs, with the exception of the replication-dependent
histone mRNAs, terminate at the 3' end with a poly(A) tail.
Replication-dependent histone mRNAs end instead in a conserved 26-nucleotide
sequence that contains a 16-nucleotide stem-loop. Formation of the 3' end of
histone mRNA occurs by endonucleolytic cleavage of pre-mRNA releasing the
mature mRNA from the chromatin template. Cleavage requires several trans-acting
factors, including a protein, the stem-loop binding protein (SLBP), which binds
the 26-nucleotide sequence; and a small nuclear RNP, U7 snRNP. There are
probably additional factors also required for cleavage. One of the functions of
the SLBP is to stabilize binding of the U7 snRNP to the histone pre-mRNA. In
the nucleus, both U7 snRNP and SLBP are present in coiled bodies, structures
that are associated with histone genes and may play a direct role in histone
pre-mRNA processing in vivo. One of the major regulatory events in the cell
cycle is regulation of histone pre-mRNA processing, which is at least partially
mediated by cell-cycle regulation of the levels of the SLBP protein.

Dominski Z, Marzluff WF.
Formation of the 3' end of histone mRNA.
Gene. 1999 Oct 18;239(1):1-14. Review.

Formation of the 3' end of histone mRNA.

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.

Dominski Z, Yang XC, Raska CS, Santiago C, Borchers CH, Duronio RJ, Marzluff WF.
3' end processing of Drosophila melanogaster histone pre-mRNAs: requirement for
phosphorylated Drosophila stem-loop binding protein and coevolution of the
histone pre-mRNA processing system.
Mol Cell Biol. 2002 Sep;22(18):6648-60.

3' end processing of Drosophila melanogaster histone pre-mRNAs: requirement for phosphorylated Drosophila stem-loop binding protein and coevolution of the histone pre-mRNA processing system.

Grzesiek, S. and Bax, A. (1992)
J. Magn. Reson. 99, 201-207.

Grzesiek, S., Anglister, J. and Bax, A. (1993)
J. Magn. Reson. B 101, 114-119.

Histone mRNA metabolism is tightly coupled to cell cycle progression and to
rates of DNA synthesis. The recent identification of several novel proteins
involved in histone gene transcription and pre-mRNA processing has shed light
on the variety of mechanisms cells employ to achieve this coupling.

Marzluff WF, Duronio RJ.
Histone mRNA expression: multiple levels of cell cycle regulation and important
developmental consequences.
Curr Opin Cell Biol. 2002 Dec;14(6):692-9. Review.

Histone mRNA expression: multiple levels of cell cycle regulation and important developmental consequences.

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.

Sanchez R, Marzluff WF.
The stem-loop binding protein is required for efficient translation of histone
mRNA in vivo and in vitro.
Mol Cell Biol. 2002 Oct;22(20):7093-104.

The stem-loop binding protein is required for efficient translation of histone mRNA in vivo and in vitro.

Replication-associated histone genes encode the only metazoan mRNAs that lack
polyA tails, ending instead in a conserved 26-nt sequence that forms a
stem-loop. Most of the regulation of mammalian histone mRNA is
posttranscriptional and mediated by this unique 3' end. Stem-loop-binding
protein (SLBP) binds to the histone mRNA 3' end and is thought to participate
in all aspects of histone mRNA metabolism, including cell cycle regulation. To
examine SLBP function genetically, we have cloned the gene encoding Drosophila
SLBP (dSLBP) by a yeast three-hybrid method and have isolated mutations in
dSLBP. dSLBP function is required both zygotically and maternally. Strong dSLBP
alleles cause zygotic lethality late in development and result in production of
stable histone mRNA that accumulates in nonreplicating cells. These histone
mRNAs are cytoplasmic and have polyadenylated 3' ends like other polymerase II
transcripts. Hypomorphic dSLBP alleles support zygotic development but cause
female sterility. Eggs from these females contain dramatically reduced levels
of histone mRNA, and mutant embryos are not able to complete the syncytial
embryonic cycles. This is in part because of a failure of chromosome
condensation at mitosis that blocks normal anaphase. These data demonstrate
that dSLBP is required in vivo for 3' end processing of histone pre-mRNA, and
that this is an essential function for development. Moreover, dSLBP-dependent
processing plays an important role in coupling histone mRNA production with the
cell cycle.

Sullivan E, Santiago C, Parker ED, Dominski Z, Yang X, Lanzotti DJ, Ingledue
TC, Marzluff WF, Duronio RJ.
Drosophila stem loop binding protein coordinates accumulation of mature histone
mRNA with cell cycle progression.
Genes Dev. 2001 Jan 15;15(2):173-87.

Drosophila stem loop binding protein coordinates accumulation of mature histone mRNA with cell cycle progression.

Complexes of different electrophoretic mobility containing the stem-loop binding
protein, a 45 kDa protein, bound to the stem-loop at the 3' end of histone
mRNA, are present in both nuclear and cytoplasmic extracts from mammalian
cells. We have determined the effect of changes in the loop, in the stem and in
the flanking sequences on the affinity of the SLBP for the 3' end of histone
mRNA. The sequence of the stem is particularly critical for SLBP binding.
Specific sequences both 5' and 3' of the stem-loop are also required for
high-affinity binding. Expanding the four base loop by one or two uridines
reduced but did not abolish SLBP binding. RNA footprinting experiments show
that the flanking sequences on both sides of the stem-loop are critical for
efficient binding, but that cleavages in the loop do not abolish binding. Thus
all three regions of the RNA sequence contribute to SLBP binding, suggesting
that the 26 nt at the 3' end of histone mRNA forms a defined tertiary structure
recognized by the SLBP.

Williams AS, Marzluff WF.
The sequence of the stem and flanking sequences at the 3' end of histone mRNA
are critical determinants for the binding of the stem-loop binding protein.
Nucleic Acids Res. 1995 Feb 25;23(4):654-62.

The sequence of the stem and flanking sequences at the 3' end of histone mRNA are critical determinants for the binding of the stem-loop binding protein.

Previous studies by Wishart et al. [Wishart, D. S., Sykes, B. D., & Richards, F.
M. (1991) J. Mol. Biol. (in press)] have demonstrated that 1H NMR chemical
shifts are strongly dependent on the character and nature of protein secondary
structure. In particular, it has been found that the 1H NMR chemical shift of
the alpha-CH proton of all 20 naturally occurring amino acids experiences an
upfield shift (with respect to the random coil value) when in a helical
configuration and a comparable downfield shift when in a beta-strand extended
configuration. On the basis of these observations, a technique is described for
rapidly and quantitatively determining the identity, extent, and location of
secondary structural elements in proteins based on the simple inspection of the
alpha-CH 1H resonance assignments. A number of examples are provided to
demonstrate both the simplicity and the accuracy of the technique. This new
method is found to be almost as accurate as the more traditional NOE-based
methods of determining secondary structure and could prove to be particularly
useful in light of the recent development of sequential assignment techniques
which are now almost NOE-independent [Ikura, M., Kay, L. E., & Bax, A. (1990)
Biochemistry 29, 4659-4667]. We suggest that this new procedure should not
necessarily be seen as a substitute to existing rigorous methods for secondary
structure determination but, rather, should be viewed as a complement to these
approaches.

Wishart DS, Sykes BD, Richards FM.
The chemical shift index: a fast and simple method for the assignment of
protein secondary structure through NMR spectroscopy.
Biochemistry. 1992 Feb 18;31(6):1647-51.

The chemical shift index: a fast and simple method for the assignment of protein secondary structure through NMR spectroscopy.

Stem-loop binding protein [Drosophila melanogaster]

replication-associated histone mRNA stem loop-binding protein [Drosophila melanogaster]

histone mRNA stem-loop binding protein [Drosophila melanogaster]

RecName: Full=Histone RNA hairpin-binding protein; AltName: Full=Histone stem-loop-binding protein [Drosophila melanogaster]