Research Interests

The splicing of nuclear pre-mRNAs is a fundamental process required for the expression of most metazoan genes. It is carried out by the spliceosome which recognizes splicing signals and catalyzes the removal of non-coding intronic sequences to assemble protein coding sequences into mature mRNA prior to export and translation. Through the differential joining of coding sequences, alternative splicing allows for the production of multiple isoforms from a single gene, therefore exponentially enriching the proteomic diversity of higher eukaryotic organisms. Consequently, the process of splicing must occur with a high degree of specificity and fidelity to ensure the appropriate expression of functional mRNAs. Defects in splicing lead to many human genetic diseases, and splicing mutations in a number of genes involved in growth control have been implicated in multiple types of cancer. Understanding the basic mechanisms of pre-mRNA splicing and splice site recognition is therefore a prerequisite to understanding the regulated expression of alternatively or constitutively spliced genes and to the understanding and, ultimately, treatment of human diseases. The Hertel laboratory focuses on the following broad research topics:

• Mechanisms of pre-mRNA splicing and the function of splicing enhancers in exon definition
• Regulation of pre-mRNA splicing and human genetic diseases
• The coupling of transcription and pre-mRNA splicing

Mechanisms of pre-mRNA splicing and the function of splicing enhancers in exon definition. Our overall goal is to understanding the molecular events that lead to alternative splice site pairing and thus, to the generation of multiple mRNA isoforms.  We have concentrated our efforts on defining when the spliceosome commits splice sites for pairing and what functional role cis-acting RNA elements play in the process of splice site recognition.  One of our model genes, the Drosophila fruitless gene, encodes a transcription factor that regulates essentially all aspects of male courtship behavior.  The use of alternative 5' splice sites generates fru isoforms that determine gender-appropriate sexual behaviors.  Alternative splicing of fruitless is regulated by the splicing activators Tra and Tra2, and depends on an exonic splicing enhancer (ESE) consisting of three 13 nucleotide repeat elements, nearly identical to those that regulate alternative sex-specific 3' splice site choice in the doublesex gene.  Doublesex has provided a powerful model system to investigate the mechanisms of enhancer-dependent 3' splice site choice.  However, little is known about enhancer dependent regulation of alternative 5’ splice sites.  We have investigated the mechanisms of alternative 5’ splice site choice by using an in vitro system in which recombinant Tra/Tra2 could activate the female-specific 5’ splice site of fruitless.  Our analysis demonstrated that splicing enhancers function similarly in activating regulated 5' and 3' splice sites.  These results suggested that exonic splicing enhancers recruit multiple spliceosomal components required for the initial recognition of 5' and 3' splice sites (A).  To substantiate this observation, we set out to determine if the stimulatory activity of an individual ESE is limited to the activation of one of the splice sites or if a single ESE simultaneously promotes the recognition of both exon/intron junctions. Our results demonstrated that an individual enhancer complex is sufficient to activate both weak splice sites (A).  Thus, ESEs recruit a complex that minimally contains factors necessary for both 3' and 5' splice site recognition.  Our data is consistent with a model in which components of the splicing machinery that define the boundaries of exon/intron junctions are assembled prior to recruitment to the exon (B).

Recent work from our laboratory has answered a very critical question that has remained elusive for almost 20 years of studying pre-mRNA splicing – when does splice site pairing occur? After definition of the exons, the spliceosome is activated by a series of sequential structural rearrangements. Formation of the first ATP-independent spliceosomal complex commits the pre-mRNA to the general splicing pathway. However, the time at which a commitment to a specific splice site choice and pairing is made has been unknown.  We were able to demonstrate that alternative splicing patterns are irreversibly chosen at a kinetic step different from the ATP-independent commitment to splicing. Splice sites become committed at the first ATP-dependent spliceosomal complex when rearrangements lock U2 snRNP onto the pre-mRNA.  Thus, commitment to the splicing pathway and commitment to splice site pairing are separate steps during spliceosomal assembly and ATP hydrolysis drives the irreversible juxtaposition of exons within the spliceosome. The results set the stage for further investigations geared towards understanding the molecular and physical events that lead to irreversible pairing.

Assembly of a splicing enhancer complex precedes splice site activation. In order to understand how alternative splice sites are recognized we must have a clear understanding of enhancer complex assembly. Crucial for productive splice site activation is the stability and specificity of interactions between splicing factors bound to the ESE and the spliceosome. In the case of dsx the splicing factors Tra, Tra2, and RBPI assemble onto the dsx ESE. Our goal is to define the architecture of this heterotrimeric splicing enhancer complex using in vitro reconstitution approaches that allows us to carefully analyze the thermodynamics and kinetics of the cooperative assembly. Of particular interest is the characterization of the protein-protein interactions within the enhancer required for assembly.

Regulation of pre-mRNA splicing and human genetic diseases. Proximal spinal muscular atrophy (SMA) is a common human genetic disease that is the leading cause of hereditary infant mortality. It is characterized by the progressive degeneration of the anterior horn stem cells of the spinal cord with consequent paralysis of the trunk and limbs. Three clinical groups of the disease (I - III) have been described based on the decreasing severity of the symptoms. SMA has been linked to deletions or mutations of the Survival of Motor Neuron (SMN) gene, which has been mapped as an inverted repeat to chromosome 5 at 5q13. Homozygous absence of the telomeric copy (SMN1) correlates with development of SMA. By contrast, alterations within the centromeric SMN gene (SMN2) do not produce any known phenotype. A genomic sequence comparison of the two genes revealed that SMN1 and SMN2 encode for the identical protein. However, three alternatively spliced transcripts generated with different efficiencies have been described for each locus. SMN1 primarily produces the full-length form of SMN, whereas differential splicing of the SMN2 pre-mRNA predominantly produces an isoform lacking exon 7 (SMND7). Comparison of SMN transcripts revealed a direct relationship between SMA and exon 7 skipping. The only critical nucleotide change between SMN1 and SMN2 affecting the inclusion of exon 7 has been pinpointed to a single C to T base difference located six nucleotides inside exon 7. This transition disrupts a putative ESE and fortuitously creates a splicing silencer element. As a consequence, exon 7 is skipped in SMN2 and SMND7 is the product of an alternative processing event. Because all individuals with SMA have retained their SMN2 allele, therapy directed towards increasing SMN2 exon 7 inclusion could provide a promising tool to lower the clinical severity of SMA. Our research has focused on developing and testing molecular strategies that redirect the SMN2 splicing pattern to produce full length, viable SMN protein. We have developed an antisense oligonucleotide-based strategy that alters the splicing pattern of the SMN2 transcript.  We demonstrated that the application of antisense oligonucleotides targeting the 3' splice site of SMN2 exon 8 efficiently tilted the balance of splice site competition in favor of exon 7 inclusion. Thus, we have a technology at hand that allows SMN2 to be turned into a true backup gene. As a natural extension of these promising results, we are now focusing on further optimizing the antisense strategy developed and on generating viral delivery vehicles appropriate for efficacy testing in SMA animal models.

The coupling of transcription and pre-mRNA splicing.  Our laboratory is investigating the proposal that processing of pre-mRNAs occurs co-transcriptionally. Using a coupled transcription/splicing assay we are analyzing the mechanisms of splice site choice and pairing of de novo synthesized pre-mRNAs. The goals are to evaluate the efficiency and fidelity of regulated splicing of pre-mRNAs that were transcribed from a variety of inducible promoters and to characterize the physical link between the transcription and splicing machinery.

UCI Faculty Profile: Klemens J. Hertel.

Selected Publications:

Sciabica, K. S., and Hertel, K.J. 2006. The splicing regulators Tra and Tra2 are unusually potent activators of pre-mRNA splicing. Nucleic Acids Res, doi: 10.1093/nar/gkl984.

Dou, Y.*, Fox-Walsh, K.L.*, Baldi, P.F., and Hertel, K.J. 2006. Genomic splice-site analysis reveals frequent alternative splicing close to the dominant splice site. RNA, 12: 2047-2056.
*Joint first authors.

Hicks, M.J., Yang, C.R., Kotlajich, M.V., and Hertel, K.J. 2006. Linking splicing to Pol II transcription stabilizes pre-mRNAs and influences splicing patterns. PLoS Biol 4: e147, 943-951.

Hicks, M.J., Lam, B.J., and Hertel, K.J. 2005. Analyzing mechanisms of alternative pre-mRNA splicing using in vitro splicing assays. Methods 37: 306-313.

Madocsai, C., Lim, S.R., Geib, T., Lam, B.J., and Hertel, K.J. 2005. Correction of SMN2 Pre-mRNA splicing by antisense U7 small nuclear RNAs. Mol Ther 12: 1013-1022.

Hertel, K.J. & Graveley, B.R., (2005) RS domains contact the pre-mRNA throughout spliceosome assembly, TIBS, 30, 115-118.

Ibrahim E.C, Schaal T.D., Hertel K.J., Reed R., & Maniatis T. (2005) Serine/arginine-rich protein-dependent suppression of exon skipping by exonic splicing enhancers, Proc. Natl. Acad. Sci. USA 102, 5002-5007.

Freund M., Hicks M.J., Konermann C., Otte M., Hertel K.J., & Schaal H. (2005) Extended base pair complementarity between U1 snRNA and the 5 splice site does not inhibit splicing in higher eukaryotes, but rather increases 5 splice site recognition, Nucleic Acids Res. 33, 5112-5119.

Fox-Walsh K.L., Dou Y., Lam B.J., Hung S., Baldi P.F., & Hertel K.J. (2005) The Architecture of Pre-mRNAs Affects Mechanisms of Splice-site pairing, Proc. Natl. Acad. Sci. USA, 102, 16176-1618.

Lim S.R., & Hertel, K.J. (2004), Commitment to splice site pairing coincides with A complex formation, Mol. Cell 15, 477-483.

List of Publications via PubMed (NIH National Library of Medicine)