1. Modification of U6 Spliceosomal RNA Is Guided by Other Small RNAs
Kazimierz T Tycowski, Zhi-Hao You, Paul J Graham, Joan A Steitz 
Molecular Cell 
Volume 2, Issue 5, Pages (November 1998)
DOI: /S (00)

2. Figure 1 Mammalian U6 snRNA Shown Paired to U4 snRNA
The sequence of U6 is conserved among mammalian species, while the sequence of U4 shown is that of human U4A (Reddy 1988). The modification pattern of U6 has been established for mouse (Harada et al. 1980) and rat (Epstein et al. 1980), while that of U4 for rat (Reddy et al. 1981) and human (Krol et al. 1981) RNAs. Modified nucleotides are specified as follows: Am, Cm, and Gm: 2′-O-methyladenosine, -cytidine, and -guanosine, respectively; m6A: N6-methyladenosine; m2G: 2-methylguanosine; mpppG, γ-methyl-phosphate-guanosine triphosphate; 2,2,7m3G, 2,2,7-tri-methylguanosine; ψ, pseudouridine. The sequences of U6 snRNA involved in base-pairing interactions during splicing are shaded. The Sm site of U4 is boxed. The C6 to U substitution in the Xenopus tropicalis U6 snRNA (Krol et al. 1987) relative to mammals is shown. Although the sequence of the X. laevis U6 has not been established, the phylogenetic conservation of U6 across the entire eukaryotic kingdom argues that it is identical to its X. tropicalis counterpart.
Molecular Cell 1998 2, DOI: ( /S (00) )

3. Figure 2 mgU6-47 and mgU6-77 RNAs Are Associated with Fibrillarin
RNAs were precipitated from nuclear sonicates of mouse L1210 cells with either Y12 anti-Sm (lanes 2) or 72B9 anti-fibrillarin (lanes 3) monoclonal antibodies and probed for mgU6-47 (A) and mgU6-77 (B) RNAs by Northern blotting. Lanes 1 contain nuclear RNA from only half as many cells as in other lanes. To control for loading, the blots were reprobed for U2 snRNA. The MspI digested pBR322 size markers were used.
Molecular Cell 1998 2, DOI: ( /S (00) )

4. Figure 3
Primary and Predicted Secondary Structures of mgU6-47 and mgU6-77 RNAs
The sequences of the mouse and C. elegans mgU6-47, as well as mouse mgU6-77, were retrieved from the GenBank database as parts of the entries deposited under accession numbers AA138581, U13642, and L36610, respectively. The sequence of Xenopus mgU6-77 has been deposited under accession number AF The 5′ and 3′ ends of the RNAs were inferred from the location of boxes C and D (Kiss-Laszlo et al. 1996). Conserved boxes C, C′, D, and D′ as well as the complementary sequences in U6 snRNA and 28S rRNA are shown. Arrows indicate residues predicted to be involved in the formation of terminal stems. The shaded residues constitute a conserved terminal loop. The nucleotides in U6 and 28S RNAs either shown (Maden 1990) or predicted to be 2′-O-methylated (only in the cases of C. elegans U6 and mouse 28S) are marked by “m.”
Molecular Cell 1998 2, DOI: ( /S (00) )

5. Figure 4
Depletion of mgU6-77 RNA Inhibits 2′-O-methylation of C77 in U6 snRNA
(A and B) Oocytes were injected with antisense mgU6-77-5′ or a nonspecific deoxyoligonucleotide as indicated at the top. Eighteen hours later, the oocytes were injected with uniformly labeled U6 snRNA; after a 7 hr incubation, they were dissected and RNA isolated from the nuclei. (A) The RNA was probed for mgU6-77 and U28 snoRNA by primer extension using a mixture of mgU6-77-3′ and U28-3′ primers. mgU6-77* indicates the mgU6-77-5′ oligonucleotide-induced 3′ degradation product of mgU6-77 RNA. (B) RNA was digested with RNase H in the presence of chimeric oligonucleotide Chim-U6. U6-5′ and U6-3′ stand for 5′ and 3′ cleavage products of U6 snRNA, respectively.
(C and D) 18 hr after mgU6-77-5′ oligonucleotide injection, the oocytes were injected with either mgU6-77 or antisense mgU6-77 transcript as indicated at the top. The rescue transcripts were injected into either the cytoplasm (lanes 5 and 6 in [C] and 3 and 4 in [D]) or the nucleus (lanes 7 and 8 in [C] and 5 and 6 in [D]). Twenty hours later, uniformly labeled U6 snRNA was injected, and after a 7 hr incubation the oocytes were dissected and RNA isolated from the nuclei. (C) Cleavage of RNA by RNase H directed by oligonucleotide Chim-U6. (D) Northern blot analysis of mgU6-77 and control U25 snoRNA. Note that nuclear injection of the mgU6-77 rescue transcript led to a 50-fold-higher accumulation of mgU6-77 RNA than the cytoplasmic injection. Both the primer extension products and U6 snRNA were resolved on 8% denaturing polyacrylamide gels.
Molecular Cell 1998 2, DOI: ( /S (00) )

6. Figure 5
The U6 Region Complementary to mgU6-77 Is Sufficient for 2′-O-methylation of the C77 Residue
(A) C-1 RNA. The underlined sequence is identical to nucleotides 70–83 of U6 snRNA containing the complement to mgU6-77 and a single flanking residue on each end. The rest of the C-1 sequence contains no more than 5 contiguous nucleotides identical to U6. The C residue corresponding to C77 in U6 is indicated by an arrow. (B) Analysis of 2′-O-methylation at the C residue corresponding to C77 in U6. Oocytes were injected with the antisense mgU6-77-5′ deoxyoligonucleotide as indicated at the top. Eighteen hours later, the oocytes were injected with uniformly labeled C-1 RNA; after a 4 hr incubation, they were dissected and RNA isolated from the nuclei. The RNA was subsequently digested with RNase H in the presence (lanes 4 and 6) or absence (lanes 3 and 5) of chimeric oligonucleotide Chim-C-1. Lanes 1 and 2 show RNase H digestion of uninjected C-1 RNA in the absence and presence of Chim-C-1 oligonucleotide, respectively. C-1* indicates a product generated by 3′ end trimming of C-1 RNA. C-1-5′ and C-1-3′ or C-1*-5′ and C-1*-3′ stand for 5′ and 3′ cleavage products of C-1 or C-1*, respectively. RNAs were resolved on 8% denaturing polyacrylamide gel.
Molecular Cell 1998 2, DOI: ( /S (00) )

7. Figure 6
Depletion of mgU6-77 RNA Inhibits 2′-O-methylation of C2970 in 28S rRNA
Oocytes were cytoplasmically injected with antisense mgU6-77-5′ (lanes 3–5 and 8–10) or a nonspecific (lanes 2 and 7) deoxyoligonucleotide. Sixteen hours later, mgU6-77 (lanes 4 and 9) or antisense mgU6-77 (lanes 5 and 10) transcripts were injected into germinal vesicles. After overnight labeling with [α-32P]UTP, RNA from both the nuclear and cytoplasmic compartments was isolated. (A) RNA was subjected to chimeric oligonucleotide Chim-28S-directed RNase H cleavage and resolved on a 1% agarose-formaldehyde gel. The 18S and 28S rRNAs and their precursors are indicated on the left, while the RNase H cleavage products are indicated at the right. Band 40S-5′ in lane 3 is stronger than that in lane 5 and the intact 40S band in other lanes because in the RNase H undigested material of lane 3 the 40S precursor was more abundant (data not shown), due to a lower level of nonspecific degradation in this sample. (B) RNA was fractionated on a 10% denaturing polyacrylamide gel and probed for mgU6-77 by Northern blotting.
Molecular Cell 1998 2, DOI: ( /S (00) )

8. Figure 7 mgU6-47 and mgU6-77 RNAs Cofractionate with Nucleoli
HeLa cells were separated into cytoplasmic and nuclear fractions and then the nuclei were further fractionated into nucleoplasm and nucleoli. RNA was isolated from each fraction, resolved on a 7% denaturing polyacrylamide gel, and probed for mgU6-47 and mgU6-77 by Northern blotting using oligonucleotide mgU6-47-5′ and mgU6-77 riboprobe, respectively. The blots were reprobed for nucleolar U25 snoRNA and nucleoplasmic U6 and U2 snRNAs using riboprobes. To avoid overloading, lane 1 contained total RNA from only half as many cells as in the other lanes. The majority of the U15 signal in lane 1 was retained at the top of the gel, most likely because of its hybridization via two predicted antisense elements to two sites in 28S rRNA (our unpublished data).
Molecular Cell 1998 2, DOI: ( /S (00) )