Chemical approaches for structure and function of RNA in postgenomic era.

In the study of cellular RNA chemistry, a major thrust of research focused upon sequence determinations for decades. Structures of snRNAs (4.5S RNA I (Alu), U1, U2, U3, U4, U5, and U6) were determined at Baylor College of Medicine, Houston, Tex, in an earlier time of pregenomic era. They show novel modifications including base methylation, sugar methylation, 5'-cap structures (types 0-III) and sequence heterogeneity. This work offered an exciting problem of posttranscriptional modification and underwent numerous significant advances through technological revolutions during pregenomic, genomic, and postgenomic eras. Presently, snRNA research is making progresses involved in enzymology of snRNA modifications, molecular evolution, mechanism of spliceosome assembly, chemical mechanism of intron removal, high-order structure of snRNA in spliceosome, and pathology of splicing. These works are destined to reach final pathway of work "Function and Structure of Spliceosome" in addition to exciting new exploitation of other noncoding RNAs in all aspects of regulatory functions.

Thermodynamic analyses of the constitutive splicing pathway for ovomucoid pre-mRNA.

The ovomucoid pre-mRNA has been folded into mini-hairpins adaptable for the RNA recognition motif (RRM) protein binding. The number of mini-hairpins were 372 for pre-mRNA and 83-86 for mature m RNA The spatial arrangements are, in average, 16 nucleotides per mini-hairpin which includes 7 nt in the stem, 5.6 nt in the loop and 3.7 nt in the inter-hairpin spacer. The constitutive splicing system of ovomucoid-pre-mRNA is characterized by preferred order of intron removal of 5/6 > 7/4 > 2/1 > 3. The 5' splice sites (5'SS), branch point sequences (BPS) and 3' splice sites (3'SS) were identified and free energies involved have been estimated in 7 splice sites. Thermodynamic barriers for splice sites from the least (| lowest | -Kcal) were 5,4, 7,6, 2,1, and 3; i.e., -18.7 Kcal, -20.2 Kcal, -21.0 Kcal, -24.0 Kcal, -25.4 Kcal, -26.4 Kcal and -28.2 Kcal respectively. These are parallel to the kinetic data of splicing order reported in the literature. As a result, the preferred order of intron removals can be described by a consideration of free energy changes involved in the spliceosomal assembly pathway. This finding is consistent with the validity of hnRNP formation mechanisms in previous reports.

A modeling study of co-transcriptional metabolism of hnRNP using FMR1 gene.

Since molecular structure of hnRNP is not available in foreseeable future, it is best to construct a working model for hnRNP structure. A geometric problem, assembly of 700 +/- 20 nucleotides with 48 proteins, is visualized by a frame work in which all the proteins participate in primary binding, followed by secondary, tertiary and quaternary binding with neighboring proteins without additional import. Thus, 40S hnRNP contains crown-like secondary structure (48 stem-loops) and appearance of 6 petal (octamers) rose-like architectures. The proteins are wrapped by RNA. Co-transcriptional folding for RNP fibril of FMR1 gene can produce 2,571 stem-loops with frequency of 1 stem-loop/15.3 nucleotides and 53 40S hnRNP beaded structure. By spliceosome driven reactions, there occurs removal of 16 separate lariated RNPs, joining 17 separate beaded exonic structures and anchoring EJC on each exon junction. Skipping exon 12 has 5'GU, 3'AG and very compact folding pattern with frequency of 1 stem-loop per 12 nucleotides in short exon length (63 nucleotides). 5'end of exon 12 contains SS (Splicing Silencer) element of UAGGU. In exons 10, 15 and 17 where both regular and alternative splice sites exist, SS (hnRNP A1 binding site) is observed at the regular splicing site. End products are mature FMR-1 mRNP, 4 species of Pri-microRNAs derived from introns 7,9,15 and 3'UTR of exon17, respectively. There may also be some other regulatory RNAs containing ALU/Line elements as well.

Structural elements of dynamic RNA strings.

Short transient secondary structures form while RNA is being transcribed, and these become the initial sites for protein anchoring. The insulin gene transcript (IGT) of chain length 1,430 nucleotides can be folded into 92 stem-loops, at an average of one stem-loop per 15.5 nucleotides (range: 9-35). The 25-hydroxyvitamin D3 1-alpha-hydroxylase gene transcript (HDHGT) of 4,825 nucleotides can fold into 274 stem-loops, at one stem-loop per 17.6 nucleotides (range: 9-45). We found no differences in transient secondary structures between the exons of IGT and HDHGT but there were significant differences between the introns. RNA chain shortening by folding ranged from 2.57 to 9.6 fold. Contraction ratios for IGT were 2.79 for minimal contraction and 7.77 for maximal contraction, and for HDHGT 2.57 and 8.80 respectively. The maximal contraction ratios but not the minimal contraction ratios differed significantly between IGT and HDHGT. This implies that initial RNP fibril formation may proceed by shared mechanisms whereas the final degree of compaction can differ in different hnRNPs. Metastable co-transcriptional folding may be necessary for "chaperones"/"match makers" to refold the RNA correctly for splicing and other maturation process. Branch point sequences are not consistent and are not included in the analysis. However, 5' and 3' splice regions have more disordered secondary structures, and 3' and 5' exon regions contain intrinsic snap-back complementarity that can bring 3' and 5' nucleotides together for joining. Upon splicing, the remaining exons undergo no change except for a few stem-loops flanking the splice sites.