James Bruzik - Academia.edu (original) (raw)

Papers by James Bruzik

Genes & Development, Jun 15, 1999

Serine/arginine-rich splicing factors (SR proteins) are substrates for serine phosphorylation tha... more Serine/arginine-rich splicing factors (SR proteins) are substrates for serine phosphorylation that can regulate SR protein function. We have observed gross changes in SR protein phosphorylation during early development coincident with major zygotic gene activation in the nematode Ascaris lumbricoides. These differences correlate with large-scale changes in SR protein activity in promoting both trans-and cis-splicing. Importantly, inactive early stage extracts can be made splicing competent on addition of later stage SR proteins. These data suggest that changes in SR protein phosphorylation have a role in the activation of pre-mRNA splicing during early development.

Splice-site selection and alternative splicing of nuclear pre-mRNAs can be controlled by splicing... more Splice-site selection and alternative splicing of nuclear pre-mRNAs can be controlled by splicing enhancers that act by promoting the activity of upstream splice sites. Here we show that RNA molecules containing a 3' splice site and enhancer sequence are efficiently spliced in trans to RNA molecules containing normally cis-spliced 5' splice sites or to normally trans-spliced spliced leader RNAs from lower eukaryotes. In addition, we show that this reaction is stimulated by (Ser + Arg)-rich splicing factors that are known to promote protein-protein interactions in the cis-splicing reaction. Thus, splicing enhancers facilitate the assembly of protein complexes on RNAs containing a 3' splice site, and this complex is sufficiently stable to functionally interact with 5' splice sites located on separate RNAs. This trans-splicing is mediated by interactions between (Ser + Arg)-rich splicing factors bound to the enhancer and general splicing factors bound to the 5' and 3' splice sites. These same interactions are likely to play a crucial role in alternative splicing and splice-site selection in cis.

Molecular Biology Reports, Feb 1, 1990

The trans-splicing reaction characterized in nematodes and in trypanosomes and their relatives in... more The trans-splicing reaction characterized in nematodes and in trypanosomes and their relatives involves the addition of a short leader exon onto the 5' end of a premRNA transcript. We have proposed (Bruzik et al., 1988) that SL RNAs, which donate the 5' leader exon and themselves assemble into snRNP particles, might perform the normal function of the U1 snRNP, making the trans-splicing reaction independent of the need for UI . To test this hypothesis, we have constructed splicing substrates that contain SL RNA sequences connected to an adenovirus 3' splice site and exon. These constructs splice well in HeLa cell nuclear extracts, even after inactivation of UI snRNPs by extensive oligonucleotide-directed RNase H degradation of the 5' end of the UI RNA. Thus, the SL RNA sequence does indeed appear to substitute for U1 during splicing in HeLa cell nuclear extracts. Studies employing mutant SL RNA-3 ' splice site constructs have further defined the precise sequence requirements for UI snRNP-independent splicing. These results will be discussed in terms of the relationship between cisand trans-splicing and the mechanism of activation of the 5" splice site in the spliceosome.

Cell, Jul 9, 1990

L. collosoma or C. elegans SL RNA sequences joined to an adenovirus intron and 3&amp;amp;... more L. collosoma or C. elegans SL RNA sequences joined to an adenovirus intron and 3&amp;amp;amp;#39; exon are spliced highly efficiently and accurately in HeLa nuclear extract. After inactivation of U1 snRNPs using RNAase H and a deoxyoligonucleotide complementary to the first 12 nucleotides of U1, splicing of SL RNA-containing constructs continues undiminished, whereas control substrates no longer splice. Since neither binding of U1 snRNPs nor inhibition of splicing is detected using anti-(U1)RNP antibodies, splicing of SL RNA-containing constructs may be entirely U1 snRNP independent. Analyses of altered L. collosoma constructs revealed that the sequence surrounding the 5&amp;amp;amp;#39; splice site is not sufficient to confer U1-independent splicing; the smallest U1-independent region identified so far retains only the first stem-loop of the SL RNA. That sequences responsible for recognition of the 5&amp;amp;amp;#39; splice site can be relocated within the splicing substrate itself reinforces the similarity between group II self-splicing and spliceosome-mediated pre-mRNA splicing.

Nucleic Acids Research, 1988

Model transcripts containing mammalian pre-rRNA sequences were incu- bated with a HeLa cell extra... more Model transcripts containing mammalian pre-rRNA sequences were incu- bated with a HeLa cell extract, digested with Tl RNase, and immunoprecipi- tated with anti-(U3)RNP or control antibodies. Two overlapping fragments derived from the 3' domain of human 28S rRNA were specifically immunoprecip- itated although transcripts which spanned the transcription initiation site, the ETS processing site, the 5' end of 18S, and

Rna-a Publication of The Rna Society, 2001

The role of exonic sequences in naturally occurring trans-splicing has not been explored in detai... more The role of exonic sequences in naturally occurring trans-splicing has not been explored in detail. Here, we have identified trans-splicing enhancers through the use of an iterative selection scheme. Several classes of enhancer sequences were identified that led to dramatic increases in trans-splicing efficiency. Two sequence families were investigated in detail. These include motifs containing the element G / C GAC G / C and also 59 splice site-like sequences. Distinct elements were tested for their ability to function as splicing enhancers and in competition experiments. In addition, discrete transacting factors were identified. This work demonstrates that splicing enhancers are able to effect a large increase in trans-splicing efficiency and that the process of exon definition is able to positively enhance trans-splicing even though the reaction itself is independent of the need for the 59 end of U1 snRNA. Due to the presence of internal introns in messages that are trans-spliced, the natural arrangement of 59 splice sites downstream of trans-splicing acceptors may lead to a general promotion of this unusual reaction.

Molecular and Cellular Biology, 2002

Proceedings of the National Academy of Sciences of the United States of America, Jan 28, 2001

Ser-Arg-rich (SR) proteins play numerous roles in spliceosome assembly and the regulation of spli... more Ser-Arg-rich (SR) proteins play numerous roles in spliceosome assembly and the regulation of splice-site selection. Whereas considerable attention has focused on the mechanistic details of SR protein activities, little is known concerning how these splicing regulators are controlled by the cell. Here we examined the subcellular localization of precursor mRNA splicing factors during early development of the nematode Ascaris lumbricoides. In the early embryo, before major zygotic gene activation, most SR proteins, along with RNA polymerase II, are localized in the cytoplasm. As development proceeds, we observe a significant decrease in the cytoplasmic levels of these factors and a concomitant increase in nuclear localization. In contrast, trimethylguanosine-capped small nuclear ribonucleoproteins are predominantly localized in the nucleus throughout this period. We previously showed that the phosphorylation state and activity of SR proteins are regulated during A. lumbricoides embryog...

RNA (New York, N.Y.), 1999

SR (ser/arg) proteins have been shown to play roles in numerous aspects of pre-mRNA splicing, inc... more SR (ser/arg) proteins have been shown to play roles in numerous aspects of pre-mRNA splicing, including modulation of alternative splicing, commitment of substrates to the splicing pathway, and splice site communication. The last of these, splice site communication, is particularly relevant to trans-splicing in which the 5' and 3' exons originate on separate molecules. The participation of SR proteins in naturally occurring, spliced leader RNA-dependent transsplicing has not been examined. Here, we have isolated SR proteins from an organism that performs both trans- and cis-splicing, the nematode Ascaris lumbricoides. To examine their activity in in vitro splicing reactions, we have also developed and characterized an SR protein-depleted whole-cell extract. When tested in this extract, the nematode SR proteins are required for both trans- and cis-splicing. In addition, the state of phosphorylation of the nematode SR proteins is critical to their activity in vitro. Interestin...

Encyclopedia of Life Sciences, 2001

Exon sequences present on separate RNA molecules can be joined by trans-splicing in trypanosomati... more Exon sequences present on separate RNA molecules can be joined by trans-splicing in trypanosomatids, Euglena, and in the nematode and trematode worms. Trans-splicing involves an interaction between a 5' splice site present in a spliced leader RNA and a 3' splice site located near the 5' end of pre-messenger RNAs. In vitro trans-splicing of artificial mammalian pre-mRNAs has been reported, but the efficiency of splicing appears to depend on sequence complementarity between the two substrates. There has been speculation that some natural pre-mRNAs can be trans-spliced in mammalian cells in vivo, but alternative interpretations have not been ruled out. Here we show that spliced leader RNAs can be accurately trans-spliced in mammalian cells in vivo and in vitro. Both nematode and mammalian 3' splice sites can function as acceptors for trans-splicing in vivo. These results reveal functional conservation in the splicing machinery between lower eukaryotes and mammals, and they directly demonstrate the potential for trans-splicing in mammalian cells.

Microbial Pathogenesis, 1996

No abstract

Cell, 1990

Journal of Biological Chemistry, 2004

Previous work demonstrated that U1 small nuclear ribonucleoprotein particle (snRNP), bound to a d... more Previous work demonstrated that U1 small nuclear ribonucleoprotein particle (snRNP), bound to a downstream 5′ splice site, can positively influence utilization of an upstream 3′ splice site via exon definition in both trans-and cis-splicing systems. ...

Rna, 2001

Biochemistry, 1987

A series of promoters with nine base-pair substitutions in the spacer D N A separating the -10 an... more A series of promoters with nine base-pair substitutions in the spacer D N A separating the -10 and -35 regions was used to demonstrate that Escherichia coli R N A polymerase is sensitive to events affecting the spacer DNA-a region not directly contacted by the enzyme. The drugs distamycin A and netropsin specifically enhanced the rate of functional complex formation at a promoter bearing a substitution of nonalternating A-T base pairs. The effect is exerted a t an early step in the R N A polymerase-promoter interaction. W e hypothesize that a drug-induced structural alteration in the spacer D N A occurs, similar to that normally resulting from R N A polymerase binding. These findings are relevant to an understanding of potential mechanisms of transcription activation.