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Biomedical subjects

T W Nilsen

Publications and source records attributed to T W Nilsen.

At least 19 recordsLinked to original sources

Dendritic nucleic acid structures.

Dendritic molecules are highly-branched arborescent structures and have found applications as chemical reagents, lubricants, contrast media for magnetic resonance, and others. Dendritic nucleic acids could be extremely useful for the development of nucleic acid diagnostics as signal amplification tools and potentially as drug (antisense) delivery vehicles. Further, due to the relatively large size of nucleic acid molecules, nucleic acid dendrimers could be readily labeled with numerous fluorescent compounds and/or protein moieties with limited steric hindrance and/or quenching. Herein, we present a physical-mathematical model of a new class of dendrimers, constructed entirely from unique nucleic acid monomers that are designed such that sequential hybridization adds successive layers of monomer in a geometric expansion of both mass and free single-stranded sequences, called arms, at the surface. The specially designed monomer is a heterodimer of two single-stranded nucleic acid oligomers possessing a central double-stranded waist and four single-stranded arms for binding. Assembly of a dendrimer is initiated from a single monomer and proceeds in layers, the first comprising four monomers, which provides 12 single-stranded arms. Thus, the second layer adds 12 monomers resulting in 36 single-stranded arms. After addition of the 6th layer, the dendrimer is comprised of 1457 monomers, of which 972 reside in the 6th layer, which possesses 2916 single-stranded arms. The accompanying mathematical description of a dendrimer's growth is generic. A natural consequence and limiting condition of the growth process we describe is a saturated solution of nucleic acid, which is, in effect, a "nucleic acid membrane".

Biopolymers

Direct analysis of nematode cis- and trans-spliceosomes: a functional role for U5 snRNA in spliced leader addition trans-splicing and the identification of novel Sm snRNPs.

Most nuclear pre-mRNAs in nematodes are processed by both cis- and trans-splicing. In trans-splicing, the 5' terminal exon, the spliced leader sequence (SL), is derived from a trans-splicing specific Sm snRNP, the SL RNP. Because U snRNPs are required cofactors for trans-splicing, and because this processing reaction proceeds via a two-step reaction pathway identical to that of cis-splicing, it has long been assumed that trans-splicing is catalyzed in a complex analogous to the cis-spliceosome. However, similarities or differences between cis- and trans-spliceosomes have not been established. In particular, the role of U5 snRNP in trans-splicing has been unclear. Here, we have used affinity selection to analyze the U snRNA constituents of nematode cis- and trans-spliceosomes. We find that U5 snRNP is an integral component of the trans-spliceosome and, using site-specific crosslinking, we show that U5 snRNP establishes specific Interactions with the SL RNA exon. We also identify two novel Sm snRNPs that are enriched in both cis- and trans-spliceosomes. Finally, we provide evidence that a SL RNP-containing multi-snRNP (SL, U4, U5, and U6 RNPs) may be a functional precursor in trans-spliceosome assembly.

Animals

Multiple requirements for nematode spliced leader RNP function in trans-splicing.

The 5' exon donor in nematode trans-splicing, the SL RNA, is a small (approximately 100 nt) RNA that resembles cis-spliceosomal U snRNAs. Extensive analyses of the RNA sequence requirements for SL RNA function have revealed four essential elements, the core Sm binding site, three nucleotides immediately downstream of this site, a region of Stem-loop II, and a 5' splice site. Although these elements are necessary and sufficient for SL RNA function in vitro, their respective roles in promoting SL RNA activity have not been elucidated. Furthermore, although it has been shown that assembly of the SL RNA into an Sm RNP is a prerequisite for function, the protein composition of the SL RNP has not been determined. Here, we have used oligoribonucleotide affinity to purify the SL RNP and find that it contains core Sm proteins as well as four specific proteins (175, 40, 30, and 28 kDa). Using in vitro assembly assays; we show that association of the 175- and 30-kDa SL-specific proteins correlates with SL RNP function in trans-splicing. Binding of these proteins depends upon the sequence of the core Sm binding site; SL RNAs containing the U1 snRNA Sm binding site assemble into Sm RNPs that contain core, but not SL-specific proteins. Furthermore, mutational and thiophosphate interference approaches reveal that both the primary nucleotide sequence and a specific phosphate oxygen within a segment of Stemloop II of the SL RNA are required for function. Finally, mutational activation of an unusual cryptic 5' splice site within the SL sequence itself suggests that U5 snRNA may play a primary role in selecting and specifying the 5' splice site in SL addition trans-splicing.

Animals

trans-splicing: an update.

5'-end maturation of messenger RNAs via acquisition of a trans-spliced leader sequence occurs in several primitive eukaryotes, some of which are parasitic. This type of trans-splicing proceeds though a two-step reaction pathway directly analogous to that of cis-splicing and like cis-splicing it requires multiple U snRNP cofactors. This minireview attempts to provide a brief synopsis of our current understanding of the evolution and biological significance of trans-splicing. Progress in deciphering the mechanism of trans-splicing, particularly as it relates to current models of cis-splicing, is also discussed.

Animals

U6 snRNA function in nuclear pre-mRNA splicing: a phosphorothioate interference analysis of the U6 phosphate backbone.

U6 snRNA is essential for and may participate in the catalysis of pre-mRNA splicing. Extensive mutational analyses in several systems have identified nucleotides essential for U6 function in splicing; however, relatively little is known regarding the role of the U6 phosphate backbone. We previously described a mutation in a nematode U6 snRNA that causes it to be used as a splicing substrate within the spliceosome. This unusual reaction has made it possible to apply modification interference analysis to U6 function. Here, we have used phosphorothioate substitution to identify pro-R oxygens throughout the U6 backbone that are necessary for the first and/or second catalytic steps of splicing. Four pro-R oxygens are important for the first step; of these only two appear to be required. One additional pro-R oxygen is uniquely required for the second step. The two pro-R oxygens critical for the first step of splicing are in the helix 1b U2/U6 interaction region and the intramolecular stem-loop of U6, respectively. A comparison of the positions of these two pro-R oxygens with those found to be critical for autocatalytic excision of a group II intron suggests a possible functional similarity between U6 snRNA and domain V of group II introns.

Animals

Most mRNAs in the nematode Ascaris lumbricoides are trans-spliced: a role for spliced leader addition in translational efficiency.

Some pre-mRNAs in nematodes are processed by trans-splicing. In this reaction, a 22-nt 5' terminal exon (the spliced leader, SL) and its associated 2,2,7-trimethylguanosine cap are acquired from a specialized Sm snRNP, the SL RNP. Although it has been evident for many years that not all nematode mRNAs contain the SL sequence, the prevalence of trans-spliced mRNAs has, with the exception of Caenorhabditis elegans, not been determined. To address this question in an organism amenable to biochemical analysis, we have prepared a message-dependent protein synthesis system from developing embryos of the parasitic nematode, Ascaris lumbricoides. Using this system, we have used both hybrid-arrest and hybrid-selection approaches to show that the vast majority (80-90%) of A. lumbricoides mRNAs contain the SL sequence and therefore are processed by trans-splicing. Furthermore, to examine the effect of SL addition on translation, we have measured levels of protein synthesis in extracts programmed with a variety of synthetic mRNAs. We find that the SL sequence itself and its associated hypermethylated cap functionally collaborate to enhance translational efficiency, presumably at the level of initiation of protein synthesis. These results indicate that trans-splicing plays a larger role in nematode gene expression than previously suspected.

Animals

Transcription of a nematode U1 small nuclear RNA in vitro. 3'-end formation requires cis-acting elements within the coding sequence.

We have used block-substitution mutagenesis and in vitro transcription assays to identify cis-acting DNA sequence elements important for initiation and 3'-end formation of a U1 small nuclear RNA (snRNA) in the parasitic nematode Ascaris lumbricoides. Efficient initiation of synthesis by RNA polymerase II requires a compact element centered approximately 50 base pairs upstream of the transcriptional start site. Surprisingly, 3'-end formation of U1 snRNAs synthesized in vitro is solely dependent upon elements within the U1 coding sequence. In all other systems studied thus far, 3'-end formation of U snRNAs requires signals present in the 3'-flanking region. We also show that sequence elements that direct 3'-end formation of the A. lumbricoides trans-spliced leader RNA function when RNA synthesis is initiated from the U1 promoter. These results indicate that 3'-end formation of U snRNAs in nematodes is mechanistically distinct from the analogous process in higher eukaryotes.

Animals

Functional reconstitution of U6 snRNA in nematode cis- and trans-splicing: U6 can serve as both a branch acceptor and a 5' exon.

Maturation of nuclear pre-mRNAs in nematodes requires both cis- and trans-splicing. Both processing pathways involve analogous two-step phosphotransfer reactions and both are dependent upon the integrity of U6 snRNA. We have developed a functional reconstitution assay to assess the U6 snRNA sequence requirements for cis- and trans-splicing. Branch formation between the splicing substrates and U6 snRNA was observed. The frequency of this event was greatly enhanced when a highly conserved sequence in U6 snRNA was altered by mutation. In cis- and trans-splicing reactions reconstituted with this mutant U6 snRNA the liberated exon of U6 proceeded through the second step of splicing using the appropriate splice acceptor sites. These results demonstrate covalent interactions between a U snRNA required for splicing and a splicing substrate, and they provide evidence for an unexpected degree of catalytic flexibility within the spliceosome.

Animals

Trans-splicing of nematode premessenger RNA.

In nematodes, many mRNAs contain a common 5' terminal 22-nt sequence. This sequence, the spliced leader (SL), is acquired from a small (approximately 100 nt) SL RNA via trans-splicing. Parallel in vitro and in vivo experiments have begun to clarify both the mechanism and biological role of trans-splicing. In vitro analysis (in cell free extracts) has shown that trans-splicing is remarkably similar to the snRNP mediated removal of intervening sequences from pre-mRNAs (cis-splicing). Additionally, this analysis has suggested a mechanism that may explain how the two substrates of trans-splicing (the SL RNA and pre-mRNA) efficiently associate with one another in the absence of sequence complementarity. In vivo experiments suggest that a major biological function of trans-splicing in nematodes may be to process polycistronic transcription units. Results obtained from the study of both parasitic and free-living species are discussed, and trans-splicing in nematodes is compared and contrasted to the analogous process in trypanosomatid protozoans.

Animals

Interaction of U6 snRNA with a sequence required for function of the nematode SL RNA in trans-splicing.

Nematode trans-spliced leader (SL) RNAs are composed of two domains, an exon [the 22-nucleotide spliced leader] and a small nuclear RNA (snRNA)-like sequence. Participation in vitro of the spliced leader RNA in trans-splicing reactions is independent of the exon sequence or size and instead depends on features contained in the snRNA-like domain of the molecule. Chemical modification interference analysis has revealed that two short sequence elements in the snRNA-like domain are necessary for SL RNA activity. These elements are sufficient for such activity because when added to a 72-nucleotide fragment of a nematode U1 snRNA, this hybrid RNA could participate in trans-splicing reactions in vitro. One of the critical sequence elements may function by base-pairing with U6 snRNA, an essential U snRNA for both cis- and trans-splicing.

Animals

Sequence requirements for maturation of the 5' terminus of human 18 S rRNA in vitro.

Creation of the mature 5' terminus of human 18 S rRNA in vitro occurs via a two-step processing reaction. In the first step, an endonucleolytic activity found in HeLa cell nucleolar extract cleaves an rRNA precursor spanning the external transcribed spacer-18 S boundary at a position 3 bases upstream from the mature 18 S terminus leaving 2',3'-cyclic phosphate, 5' hydroxyl termini. In the second step, a nucleolytic activity(s) found in HeLa cell cytoplasmic extract removes the 3 extra bases and creates the authentic 5'-phosphorylated terminus of 18 S rRNA. Here we have examined the sequence requirements for the trimming reaction. The trimming activity(s), in addition to requiring a 5' hydroxyl terminus, prefers the naturally occurring adenosine as the 5'-terminal base. By a combination of deletion, site-directed mutagenesis, and chemical modification interference approaches we have also identified a region of 18 S rRNA spanning bases +6 to +25 (with respect to the mature 5' end) which comprises a critical recognition sequence for the trimming activity(s).

Base Sequence

Antigenicity of a protective recombinant filarial protein in human bancroftian filariasis.

A 92-kDa fusion protein that encodes amino acids 1-479 of a 62-kDa Brugia malayi antigen induces resistance to microfilariae in mice. The antigenicity of this recombinant protein was explored in asymptomatic residents of Wuchereria bancrofti-endemic areas of Papua New Guinea and Egypt. Unlike sera from individuals in nonendemic areas, sera from residents of endemic areas contained IgG3 antibodies (up to dilution 1:1280) reactive with the fusion protein. There was little or no recombinant antigen-specific IgG1, IgG2, IgG4, or IgE. The mean level of IgG3 antibodies to the amino acid 1-479 construct in sera of putatively "immune" adult Papua New Guineans and children in whom microfilaremia was < 4 parasites/mL of blood was not significantly different (P < .05) from those with > 1000 parasites/mL. These data indicate that the filarial antigen corresponding to the recombinant protein is highly immunogenic in naturally infected children and adults and that the isotype and magnitude of antibody reactivity with it do not correlate with the microfilaremic status of asymptomatic persons.

Adolescent

Trans-splicing in protozoa and helminths.

Trans-splicing is defined as the process whereby exons derived from two separately transcribed RNAs are joined together. In one type of trans-splicing, nuclear pre-mRNAs acquire their 5' terminal exon (the spliced leader) from a small spliced leader RNA (SL RNA) via an RNA processing reaction that is directly analagous to the removal of intervening sequences (cis-splicing). Such leader-addition by trans-splicing has been extensively studied in trypanosomatid protozoans and in nematodes. This review summarizes recent advances in research on trans-splicing in these two systems. Progress in elucidating functionally significant sequence elements within SL RNAs and progress in understanding the mechanism and biological role of trans-splicing is discussed.

Animals