Cell Biology of the snRNP Particle

Garner

Biochemical and Biophysical Research Communications

et al. 2007

Nanosecond electric pulses generate nanopores in the interior membranes of cells and modulate cellular functions. Here, we used confocal microscopy and flow cytometry to observe Smith antigen antibody (Y12) binding to nuclear speckles, known as small nuclear ribonucleoprotein particles (snRNPs) or intrachromatin granule clusters (IGCs), in Jurkat cells following one or five 10 ns, 150 kV/cm pulses. Using confocal microscopy and flow cytometry, we observed changes in nuclear speckle labeling that suggested a disruption of pre-messenger RNA splicing mechanisms. Pulse exposure increased the nuclear speckled substructures by 2.5-fold above basal levels while the propidium iodide (PI) uptake in pulsed cells was unchanged. The resulting nuclear speckle changes were also cell cycle dependent. These findings suggest that 10 ns pulses directly influenced nuclear processes, such as the changes in the nuclear RNA-protein complexes. Published by Elsevier Inc.Keywords: Nucleus; Nuclear speckles; Small nuclear ribonucleoprotein particles/intrachromatin granule clusters; Intracellular electromanipulation; Nanosecond electric pulsesWe have been exploring a new nanosecond (ns) time domain in cell biology. When cells are exposed to intense (50-300 kV/cm) pulsed electric fields (PEFs) with rise times faster than the time it takes for charges on the plasma membrane to redistribute, the electric field can penetrate into the interior of the cell and organelles. If these fields exceed the breakdown threshold of these membranes, nanopores will form in the organelle membranes [1,2]. To better understand the mechanism involved, mathematical models for the cellular electropermeabilization due to nsPEFs have been developed [3,4]. The resulting changes in cellular structures and functions differ significantly from traditional electroporation [microseconod to millisecond pulsed electric fields (PEFs)] [1,5,6]. Increased permeabilization of the plasma membrane, typically demonstrated by measuring propidium iodide (PI) uptake, due to nsPEFs was delayed compared to traditional electroporation [5,6], indicating that it was an indirect effect. These nsPEFs 0006-291X/$ -see front matter Published by Elsevier Inc.

Section: Discussionmentioning

confidence: 99%

Nanosecond electric pulses penetrate the nucleus and enhance speckle formation

Garner

Biochemical and Biophysical Research Communications

et al. 2007

“…According to their abundance in vertebrates, the latter are divided into major snRNPs (Ul -U6) that are present in 2 x 105-1 x 106 copies per cell and the -100-fold less abundant minor snRNPs (U7-U14; Reddy and Busch, 1988;Zieve and Sauterer, 1990). The genes encoding the RNA components of the major snRNPs are organized as clustered multigene families (reviewed in Reddy and Singh, 1991) with -30 functional gene copies for Ul in mammals.…”

Section: Introductionmentioning

confidence: 99%

The low abundance of U7 snRNA is partly determined by its Sm binding site.

1993

In transient expression studies after DNA transfection of HeLa cells, the mouse U7 gene produces only -30% of the RNA produced by a mouse Ulb gene. This difference persists even when the transfected genes have all their 5' and 3' flanking sequences exchanged suggesting a post-transcriptional effect. When the special U7 Sm binding site is mutated to a consensus derived from the major snRNAs (Sm-opt), the U7 RNA level increases 4-to 5-fold, whereas no RNA is detected from a U7 gene with a non-functional Sm binding site (Smmut). Moreover, Ulb genes with the U7 Sm binding site yield reduced RNA levels. The Sm-opt site also alters the cellular behaviour of the corresponding U7 snRNA. It accumulates to a higher level in the nucleus than wild type U7 RNA, and is better immunoprecipitable with anti-Sm antibodies. Injection experiments in Xenopus oocytes indicate that the U7 genes with either Sm-opt or Sm-mut sites produce similar amounts of RNA as wild type U7, but that they differ in opposing ways in the processing of precursors to mature size U7 snRNA and in nuclear accumulation. However, in reconstitution experiments using Xenopus oocytes, we show that U7 Sm-opt RNA, despite its efficient nuclear accumulation, is not active in 3' processing of histone pre-mRNA, whereas wild type U7 RNA is assembled into functional snRNPs, which correctly process histone pre-mRNA substrate. This suggests a functional importance of the special U7 Sm sequence.

Proc. Natl. Acad. Sci. U.S.A.

“…In contrast, assembly and maintenance of snRNPs that participate in mRNA splicing is known to require ATP (25). Thus, it is possible that ATP in the T. cruzi capping reaction is required for assembly or stabilization of the putative snRNPs containing the substrate RNAs and the capping enzyme.…”

Section: Resultsmentioning

confidence: 93%

RNA-protein complexes mediate in vitro capping of the spliced-leader primary transcript and U-RNAs in Trypanosoma cruzi.

Zwierzynski¹,

Buck²

1991

A 39-nucleotide spliced leader (SL) is joined to the 5' ends of trypanosome mRNAs in a bimolecular or trans-splicing process. The SL in Trypanosoma cruzi is transcribed as an lO-nucleotide RNA (SL-RNA or SL primary transcript) bearing the 39-nucleotide SL at the 5' end. The SL-RNA is 5' capped by a guanylyltransferase activity prior to trans-splicing and trypanosome mRNAs thus obtain their mature caps from the SL by trans-splicing. We have previously characterized a guanylyltransferase activity from T. cruzi nuclear extracts and shown that this capping activity has an unusual ATP dependence and an apparent specificity for the SL-RNA and U-RNAs. Herein, we show that the capping activity sediments as a 12-15S particle during velocity sedimentation in glycerol gradients and fractionates as a >150-kDa particle during large-pore gel filtration chromatography. Moreover, the endogenous substrate RNAs-the SL-RNA and U-RNAs-consistently copurify with the capping activity, suggesting that the activity and the substrates form a ribonucleoprotein particle. The capping activity and substrate RNAs are not dissociated in isopycnic Cs2SO4 gradients and band at a density expected for an RNA-protein complex, confirming the existence of ribonucleoprotein particles bearing both the activity and its substrate RNAs. Finally, we partially purified these ribonucleoprotein particles and showed that the capping activity remains ATP dependent and highly specific for the SL-RNA and the U-RNAs. These observations are consistent with the hypothesis that one of the functions of trans-splicing is for mRNA capping. mRNA maturation in the kinetoplastid protozoa requires an unusual trans-splicing process in which a 39-nucleotide leader, the spliced leader (SL), from a small abundant transcript, the SL primary transcript (SL-RNA), is ligated to the 5' ends of structural gene primary transcripts (1). The SL-RNA obtains an unusual 5' cap that is transferred to the mature mRNA with the SL (2-5). In other eukaryotes, mRNA primary transcripts are capped shortly after transcription initiation by guanylyltransferases (GTases) that are probably associated with the RNA polymerase II (Pol II) initiation complex in a reaction requiring a 5'-tri-or -diphosphate substrate RNA (6). Although capping is thought to be mediated by the Pol II transcription initiation complex (6), soluble partially purified GTases lack substrate specificity and require only the energy from cleavage of the GTP substrate for activity. In the kinetoplastid protozoa, there is no evidence that mRNA primary transcripts are capped, many genes are transcribed as "multicistronic" RNAs that are processed into monocistronic mRNAs (cf. refs. 7 and 8), and some protein coding genes may not be transcribed by a normal a-amanitin-sensitive Pol 11 (9-11). Thus, it has been proposed that trans-splicing evolved in the kinetoplastid protozoa as a means of capping protein coding mRNAs (12).We have established (13) nuclear extracts from Trypanosoma cruzi that cap the SL-RNA and at least two U-RNAs...