Abundant nuclear ribonucleoprotein form of CAD RNA.

Abstract

Previous studies of large nuclear ribonucleoproteins (RNPs) containing mRNA and precursors of mRNA have been hampered by the heterogeneity of the material. Any one molecular species of RNA is generally present at a level of less than 1% of the total. Biochemical information about large nuclear RNPs is therefore limited to bulk properties, for the most part derived from analysis of a breakdown product of the RNP that sediments at about 30S in a sucrose gradient. This 30S complex, made up of a fragment of RNA and a set of basic proteins, is regarded as a subunit of the native structure (12,13,22).In the studies reported here, we have investigated cells containing amplified genes as a source of an abundant large nuclear RNP, with a view to the possible isolation and analysis of homogeneous material. We have used a mutant, simian virus 40 (SV40)-transformed Syrian hamster cell line in which the gene for a multifunctional enzyme abbreviated CAD (for carbamoyl-phosphate synthetase, aspartate transcarbamylase, dihydro-orotase) is amplified about 200-fold (7,11,16,27). The levels of CAD mRNA and protein show a corresponding increase, CAD mRNA making up about 1% of the cytoplasmic polyadenylated RNA (16,27). One of our first objectives was to determine whether CAD sequences are similarly enriched in nuclear RNA.A further feature of the CAD gene pertinent to studies of nuclear RNP is its size and complexity. The structure of the gene indicates that the primary transcript is 25 kilobases (kb) in length and contains 37 or more intervening sequences (17). Hybridization analysis reveals a mature mRNA of about 7.9 kb (16, 27). The great length of these molecules heightens interest in the mechanism of their packaging, processing, and transport and at the same time poses difficulties for biochemical studies, for example, the problem of instability due to a large target size for RNase. were grown in culture as described previously (24).Nuclear and cytoplasmic fractions. (i) Procedure 1. Approximately 108 cells scraped from subconfluent plates were washed three times with ice-cold 137 mM NaCI-8 mM Na2HPO4-1.5 mM KH2PO4-2.5 mM KCI, allowed to swell for 10 min in 2 ml of 10 mM Tris-hydrochloride -3 mM MgCl2 (pH 7.5) (TM), and lysed with 10 strokes of a Dounce homogenizer (B pestle) at 4°C. Nuclei were pelleted and washed, once with 1 ml of TM containing 0.5% Triton X-100 and once with 1 ml of TM. The postnuclear supernatant and detergent wash were combined and designated the cytoplasmic fraction.(ii) Procedure 2. Subconfluent plates were washed with 125 mM KCI-30 mM Tris-hydrochloride (pH 7.5;) mM magnesium acetate-1 mM 2-mercaptoethanol-2 mM ribonucleoside vanadyl complex (2)-0.15 mM spermine-0.05 mM spermidine at 4°C, and cells scraped from the plates were washed twice with the same buffer. Approximately 108 cells were allowed to swell for 10 min in 2.5 ml of swelling buffer (same as wash buffer except the KCl concentration was 10 mM), lysed with 20 strokes of a Dounce homogenizer (B pestle), overlaid on an equal volume of swellin...

RNA editing activity is associated with splicing factors in lnRNP particles: The nuclear pre-mRNA processing machinery

“…The full-length 150-kDa form of ADAR1 was found in a broad peak around the 200S region of the gradient (Fig. 1A Upper), where specific nuclear pre-mRNAs have been shown to sediment (19,(38)(39)(40). The more abundant shorter (110 kDa) form of ADAR1 had a broader distribution, with significant level sedimenting at the 200S and 70S regions of the gradient ( Fig.…”

Dual function of C/D box small nucleolar RNAs in rRNA modification and alternative pre-mRNA splicing

“…In previous experiments, fractions corresponding to the analogous positions in the gradient were enriched for the five spliceosomal U snRNAs (37,(39)(40)(41) (Fig. S1D), various pre-mRNAs (36,42,43), and regulatory splicing factors (41,44,45) (Fig. 1E) and contained active spliceosomes (37).…”

Phosphorylated Ser/Arg-rich proteins: limiting factors in the assembly of 200S large nuclear ribonucleoprotein particles.

“…This should not be surprising, because the splicing machinery of a living cell is expected to package and process multi-intronic pre-mRNAs, rather than the mono-intronic pre-mRNAs, which are typically used to assemble spliceosomes in vitro. Indeed, we have shown that several specific nuclear pre-mRNAs and their splicing products, as well as the general population of nuclear poly(A) + RNA, are packaged in compact large nuclear RNP (lnRNP) particles that invariably sediment at the 200S region in sucrose gradients (31)(32)(33)(34) …”