The structure of nuclear pre-mRNA. VII. The hybridization and renaturation properties of double-stranded sequences of nuclear pre-mRNA]

Some properties of the double-stranded regions of pre-mRNA are discribed. 1. The double-stranded regions contain approximately 80 base pairs. 2. The material contains the heterogeneous populations of sequences and some homogenous material which renatures with a COT1/2 value of (1.5-3) X 10(-4). 3. Identical sequences of fast-renaturing "hairpins" may be found in various tissues. 4. Double-stranded RNA and mRNA have some sequences complementary to each other. These results consistent with the view that the hairpin sequences may act as specific recognition sites for ribonucleases involved in processing of pre-mRNA.     

Structure of nuclear pre-mRNA. VI. "Reversed repeats" in animal DNA and their hybridization with double-stranded regions of pre-mRNA.

About 2% of mouse DNA reassociates at extremely low Cot values (10-7-10-6 mole-liter-1-sec). This DNA fraction was isolated with the aid of nuclease S1 and purified by chromatography on hydroxyapatite. The study of this fraction suggests that it has a structure of hairpin type. The DNA of the hairpins can hybridize with sequences forming the double-stranded regions in pre-mRNA. Probably at least some of the DNA of the hairpins, described as "reversed repeating sequences" of DNA, is transcribed with the formation of structures of hairpin type in pre-mRNA.     

Structure of nuclear pre-mRNA. IX. Hybridization of double-stranded portions of pre-mRNA with excess mRNA

The hybridization of double-stranded regions of pre-mRNA from mouse Ehrlich ascites carcinoma cells, rabbit bone marrow cells and primary culture of rabbit kidney cells with an excess of total poly(A)+-mRNA of mouse or rabbit globin mRNA respectively was studied. The hybrids were detected as RNAase-stable acid precipitable material or by adsorbtion of the hybrid complexes of poly(U)-sepharose. The sizes of the hybrid complementary sequences and their thermal stability were estimated.     

Direct demonstration of a complementarity between mRNA and double-stranded sequences of pre-mRNA

The total poly(A)-containing mRNA from mouse liver or Ehrlich ascites carcinoma cells was annealed with denatured ds RNA prepared from heavy nuclear ³H-labeled pre-mRNA of the same tissue. The hybrids formed were detected by binding of complexes to poly(U)-Sepharose columns through the poly(A) of mRNA. With this technique, about 30% of labeled ds RNA was bound to poly(U)-Sepharose after annealing it with an mRNA excess. The proportion of hybrid material detected by RNase treatment was two to three times lower than that obtained by poly(U)-Sepharose binding. The length of the RNase-stable acid precipitable hybrid material consisted of heterogeneous sequences of 10–100 nucleotides long when cytoplasmic, and 10–60 nucleotides long when polysomal mRNA was used in the hybridization reaction. The results obtained show that at least some of the mRNA molecules contain sequences complementary to one of the branches of the pre-mRNA hairpins. These results are compatible with the idea that the hairpin-like sequences in pre-mRNA are localized between mRNA and the non-informative part of the precursor molecule.     

Long double-stranded sequences (dsRNA-B) of nuclear pre-mRNA consist of a few highly abundant classes of sequences: evidence from DNA cloning experiments.

DNA preparations from about hundred randomly selected clones containing mouse DNA fragments were screened for the existence of sequences complementary to long double-stranded regions of pre-mRNA able to snap back after melting (dsRNA-B). Many clones containing such sequences were found. The cloned sequences can be subdivided into three groups: (1) those complementary to about a half (at least to 30-40%) of the total dsRNA, designated as sequences B1; (2) those complementary to a part of sequence B1; and (3) sequences complementary to about a quarter (at least to 15%) of the total dsRNA referred to as sequence B2. The size of DNA sequence complementary to dsRNA is about 400 base pairs.Melting experiments with hybrids show that the members of B1 family are very similar if not identical, while the divergence among B2 sequences is higher, but still the number of substitutions does not exceed 9% of bases. Thus, the major part of dsRNA-B consists of a small number of highly abundant sequences as was suggested earlier on the basis of renaturation kinetics /1-3/. Sequences B1 and B2 are represented by many copies in the mouse genome and in pre-mRNA, and many of them probably do not form hairpin-like structures.     

Globin mRNA contains a sequence complementary to double-stranded region of nuclear pre-mRNA.

Melted ds RNA isolated from rabbit bone marrow pre-mRNA was hybridized with excess of globin mRNA which was prepared from rabbit reticulocytes. 7-9% of ds sequences became RNAase-stable and about 30% of the sequences could be bound to poly(U)-Sepharose through poly (A) of mRNA. The size of RNAase-stable hybrid is about 30 nucleotides, that is one fourth of the length of one strand of the ds RNA.     

Sequences hybridizing to mRNA, oligo(dT) and dsRNA from pre-mRNA are contiguous in the cloned mouse DNA fragments.

Fragments from the DNA of mouse embryos produced by restriction endonucleases HindIII were cloned in pBR322 plasmid and examined for the ability to hybridize in situ with [32P] labeled cDNA synthesized from the polysomal poly(A)+mRNA template. Several of the selected clones were examined for the presence of specific sequences inside the cloned mouse DNA fragments by the blotting procedure of southern [1]. The data obtained indicate that the majority of the cloned mouse DNA fragments contained sequences hybridizing with cDNA, oligo(dT) and double-stranded regions from pre-mRNA. The results of hybridization experiments and double digestion with HindIII+HaeIII endonucleases provide evidence that these sequences could be contiguous in the given restriction DNA fragments.     

Secondary structure of pre-mRNA in nuclear ribonucleoprotein particles

The secondary structure of pre-mRNA in the specific nuclear ribonucleoprotein particles was investigated. After ribonuclease treatment of nuclear particles the majority of double-stranded RNA sequences was conserved. The in vivo existence of hairpin-like RNA structures is discussed.     

Cleavage of pre-mRNA sequences by ribonucleases bound to nuclear RNP particles of rat liver

The 30S nuclear RNP particles from rat liver have been shown to split the double-stranded- (ds) and single-stranded (ss) sequences of nuclear pre-mRNA. Experiments performed in vitro have demonstrated that 1) a 5-exonuclease and an endonuclease specific for double-stranded pre-mRNA sequences exist in the 30S pre-mRNP particles; 2) in dsRNA monophosphorylated 5-termini arose in the course of incubation with 30S RNP and most of the products remained double-stranded. The analysis of terminal pNp nucleotides revealed a relatively high ratio of pPyp in the cleaved dsRNA, whereas the nucleosides in 5-terminal pNp of ssRNA showed nearly random distribution. Our results provide a possible explanation for the appearance of pNp termini during the processing of nuclear pre-mRNA of mammalian cells.     

Autodegradation of pre-mRNA containing nuclear ribo-nucleoprotein particles. The effect of autodegradation on the double-stranded RNA sequences and on the protein composition of particles

The quantitative changes of double-stranded RNA components of nuclear ribonucleo-protein particles containing pre-mRNA was investigated in the course of incubation of particles at 37 ° C. The incubation of purified nuclear particles revealed the fragmentation of long double-stranded RNA sequences into shorter stretches. The presence of nuclear sap in the incubation mixture resulted in degradation of the double-stranded RNAs into acid soluble products. Autodegradation and/or ribonuclease treatment of nuclear RNP particles is accompanied by quantitative changes in the minor protein constituents of informofer.     

Secondary structure of nuclear precursors of the informational RNA (pre-mRNA)]

The secondary structure of pre-mRNA species from mouse Ehrlich ascites carcinoma cells extracted with phenol at the temperatures either 55-65 degrees C or 65-85 degrees C was investigated. A fraction of the double helical regions of pre-mRNA was estimated by two methods: a) by recording of melting curves; b) by measuring fluorescence lifetimes of acridine orange dye adsorbed on the nucleic acid. This fraction was about 64-68%. Further lowering of ionic strength down to 0.024 results in 10-15% decrease in the fraction of double regions. Both lowering of ionic strength and increase of the temperature up to 50-55 degrees C results in despiralisation of pre-mRNA regions which contain more than 70% of AU-nucleotide pairs. Only regions containing mainly GC-nucleotide pairs remain double-stranded under heating to temperatures above 50 degrees C. These facts were established on the basis of studies of acriflavin dye complexes with pre-mRNA.     

Frequency distribution of mRNA and pre-mRNA in growing and differentiated Friend cells.

The frequency distribution of poly(A)+-mRNA in growing and in differentiated Friend cells has been measured by mRNA-cDNA hybridization and their differences established by heterologous hybridization of mRNA of one type and cDNA of the other. It was shown that induction of Friend cells involves an increase in abundance of a small number of mRNAs, while no specific pattern of messenger disappearance could be detected. The frequency distribution of pre-mRNA was determined by hybridizing nuclear RNA with the cDNA probes complementary to mRNA. In uninduced Friend cells, it was shown that most precursor messenger sequences are present at a single frequency of about 3 molecules per nucleus, independently of their final frequency in polysomal mRNA. In induced Friend cells, the frequency distribution of pre-mRNA is more heterogeneous and correlated to some extent with the corresponding mRNA frequency distribution.     

Restoration of correct splicing of thalassemic beta-globin pre-mRNA by modified U1 snRNAs

The T-->G mutation at nucleotide 705 in the second intron of the beta-globin gene creates an aberrant 5' splice site and activates a 3' cryptic splice site upstream from the mutation. As a result, the IVS2-705 pre-mRNA is spliced via the aberrant splice sites leading to a deficiency of beta-globin mRNA and protein and to the genetic blood disorder thalassemia. We have shown previously that in cell culture models of thalassemia, aberrant splicing of beta-thalassemic IVS2-705 pre-mRNA was permanently corrected by a modified murine U7 snRNA that incorporated sequences antisense to the splice sites activated by the mutation. To explore the possibility of using other snRNAs as vectors for antisense sequences, U1 snRNA was modified in a similar manner. Replacement of the U1 9-nucleotide 5' splice site recognition sequence with nucleotides complementary to the aberrant 5' splice site failed to correct splicing of IVS2-705 pre-mRNA. In contrast, U1 snRNA targeted to the cryptic 3' splice site was effective. A hybrid with a modified U7 snRNA gene under the control of the U1 promoter and terminator sequences resulted in the highest levels of correction (up to 70%) in transiently and stably transfected target cells.