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Haihong Shen

Publications and source records attributed to Haihong Shen.

7 recordsLinked to original sources
PubMed2006

Structural basis for polypyrimidine tract recognition by the essential pre-mRNA splicing factor U2AF65.

The essential pre-mRNA splicing factor, U2AF(65), guides the early stages of splice site choice by recognizing a polypyrimidine (Py) tract consensus sequence near the 3' splice site. Since Py tracts are relatively poorly conserved in higher eukaryotes, U2AF(65) is faced with the problem of specifying uridine-rich sequences, yet tolerating a variety of nucleotide substitutions found in natural Py tracts. To better understand these apparently contradictory RNA binding characteristics, the X-ray structure of the U2AF(65) RNA binding domain bound to a Py tract composed of seven uridines has been determined at 2.5 A resolution. Specific hydrogen bonds between U2AF(65) and the uracil bases provide an explanation for polyuridine recognition. Flexible side chains and bound water molecules form the majority of the base contacts and potentially could rearrange when the U2AF(65) structure adapts to different Py tract sequences. The energetic importance of conserved residues for Py tract binding is established by analysis of site-directed mutant U2AF(65) proteins using surface plasmon resonance.

Amino Acid Sequence↗
PubMed2006

RS domains contact splicing signals and promote splicing by a common mechanism in yeast through humans.

Serine-arginine (SR) proteins are general metazoan splicing factors that contain an essential arginine-serine-rich (RS) domain. We have previously found that mammalian spliceosome assembly involves a series of sequential interactions between RS domains and two splicing signals: the branchpoint and the 5' splice site. Here we study how RS domains are directed to specifically contact splicing signals, and how this interaction promotes splicing. The yeast Saccharomyces cerevisiae lacks SR proteins. However, we show that tethering a mammalian RS domain to a yeast actin pre-mRNA rescues splicing of certain branchpoint or 5' splice site mutants in which U snRNA base-pairing has been decreased. Conversely, on a mammalian pre-mRNA, a normally essential SR protein becomes dispensable when the complementarity of a splicing signal to a U snRNA is increased. We find that in the absence of other splicing factors an RS domain tethered to a pre-mRNA selectively contacts a double-stranded RNA region and enhances RNA-RNA base-pairing. Significantly, all of these activities require phosphorylation of the RS domain. Based on these results, we propose that RS domains selectively contact splicing signals because, due to transient U snRNA base-pairing, they are partially double-stranded. The RS domain-splicing signal interaction, in turn, promotes (or stabilizes) base-pairing between the U snRNA and pre-mRNA substrate, thereby enhancing splicing. Our results reveal a common mechanism of RS domain function in yeast through humans.

Arginine↗
PubMed2005

Cell motility is controlled by SF2/ASF through alternative splicing of the Ron protooncogene.

Ron, the tyrosine kinase receptor for the Macrophage-stimulating protein, is involved in cell dissociation, motility, and matrix invasion. DeltaRon, a constitutively active isoform that confers increased motility to expressing cells, is generated through the skipping of exon 11. We show that abnormal accumulation of DeltaRon mRNA occurs in breast and colon tumors. Skipping of exon 11 is controlled by a silencer and an enhancer of splicing located in the constitutive exon 12. The strength of the enhancer parallels the relative abundance of DeltaRon mRNA and depends on a sequence directly bound by splicing factor SF2/ASF. Overexpression and RNAi experiments demonstrate that SF2/ASF, by controlling the production of DeltaRon, activates epithelial to mesenchymal transition leading to cell locomotion. The effect of SF2/ASF overexpression is reverted by specific knockdown of DeltaRon mRNA. This demonstrates a direct link between SF2/ASF-regulated splicing and cell motility, an activity important for embryogenesis, tissue formation, and tumor metastasis.

Alternative Splicing↗
PubMed2004

A pathway of sequential arginine-serine-rich domain-splicing signal interactions during mammalian spliceosome assembly.

Serine-arginine (SR) proteins are general splicing factors and can function through binding to exonic splicing enhancers (ESEs). SR proteins and several other mammalian splicing factors contain an arginine-serine-rich (RS) domain required to promote splicing. We have recently found that the ESE bound RS domain functions by contacting the branchpoint. Here, we perform RNA-protein crosslinking experiments to show that the branchpoint is sequentially contacted first in complex E by the RS domain of the essential splicing factor U2AF(65) and then in the prespliceosome by the ESE bound RS domain. Although the ESE bound RS domain can promote formation of the prespliceosome, at least one additional SR protein is required for complete spliceosome assembly. We show that the RS domain of this additional SR protein contacts the 5' splice site specifically in the mature spliceosome. We propose that direct contact with splicing signals is a general mechanism by which RS domains promote splicing.

Arginine↗
PubMed2004

Arginine-serine-rich domains bound at splicing enhancers contact the branchpoint to promote prespliceosome assembly.

Exonic splicing enhancers (ESEs) are required for splicing of certain pre-mRNAs and function by providing binding sites for serine-arginine (SR) proteins, which contain an arginine-serine-rich (RS) domain. How an RS domain bound at the ESE promotes splicing is poorly understood. We have developed an RNA-protein crosslinking procedure to identify the target of the ESE-bound RS domain. Using this approach, we show that the ESE-bound RS domain specifically contacts the pre-mRNA branchpoint. The interaction between the ESE-bound RS domain and the branchpoint occurs in the prespliceosome and is dependent upon the same splicing signals, biochemical factors, and reaction conditions required to support prespliceosome assembly. Analysis of RS domain mutants demonstrates that the ability to interact with the branchpoint, to promote prespliceosome assembly, and to support splicing are related activities. We conclude that the ESE-bound RS domain functions by contacting the branchpoint to promote prespliceosome assembly.

Arginine↗
PubMed2004

A single polypyrimidine tract binding protein (PTB) binding site mediates splicing inhibition at mouse IgM exons M1 and M2.

Splicing of mouse immunoglobulin (IgM) exons M1 and M2 is directed by two juxtaposed regulatory elements, an enhancer and an inhibitor, located within the M2 exon. A primary function of the enhancer is to counteract the inhibitor, allowing splicing to occur. Here we show that the inhibitor contains two binding sites for polypyrimidine tract binding protein (PTB). Mutational analysis indicates that only one of these sites is necessary and sufficient to direct splicing inhibition both in vitro and in vivo. We demonstrate that the difference in activity of the two sites is explained by proximity to the intron. We further show that the presence of the enhancer results in the disruption of the PTB-inhibitor interaction, enabling splicing to occur. In the absence of the enhancer, splicing can be artificially activated by immuno-inhibition of PTB. Collectively, our results indicate that a single PTB binding site can function as an inhibitor that regulates alternative splicing both in vitro and in vivo.

Alternative Splicing↗