Functional Reconstitution of U6 snRNA in Nematode Cis- and Trans-Splicing: U6 Can Serve as Both a Branch Acceptor and a 5' Exon

Cell 75:1049-59, Dec. 17, 1993

Introduction

1. The primary transcripts of most eukaryotic structural genes (pre-mRNA) contain

intervening sequences that are removed by RNA splicing. It is classified two major

types of splicing: Cis- and Trans-splicing by the sources of substrate of splicing.

2. Cis-splicing

A. Two exons to be joined are encoded as part of a single molecule.

B. Intron removal requires the participation of five small nuclear ribonucleoprotein particles (snRNPs) (U1,U2,U4.U5,. and U6) and a large number of proteins as yet undetermined.

C. Cis-splicing proceeds through two successive transesterification reactions within spliceosome.

3. Trans-splicing

A. Spliced-leader (SL) RNA addition trans-splicing occurs in a variety of lower eukaryotes, including the trypanosomatid protozoans and nematodes.

B. Pre-mRNAs acquire their 5' terminal exon from a small RNA (the SL RNA) that shares features in common with spliceosomal U snRNAs. i.e. join two exons from different molecules.

C. Trans-splicing proceeds through a two-step reaction pathway that generates products and intermediates directly analogous to those produced in the two- step cis-splicing reaction.

However, trans-splicing may not require the entire complement of cis- spliceosomal U snRNA.

4. U6 snRNA

A. Of the U snRNAs required for splicing, U6 snRNA is by far the most conserved. Many evidences indicate that U6 plays multiple roles in the splicing pathway.

B. In the yeast and vertebrate cis-splicing systems have identified nucleotide critical for the first and /or second catalytic steps of splicing. There is an intimate association between U6 snRNA and intron sequences near the 5' splice site.

C. U6 snRNA is central to and may participate in the catalysis of splicing.

Specific Aims:

In this paper, they used a functional reconstitution system and mutagenesis to assess the role of U6 snRNA in nematode cis- and trans-splicing.

Summary

A. Mutation of a highly conserved sequence in the central domain of U6 immediately 5' of a region in U6 causes U6 to be used as a splicing substrate in both cis-and trans-spliceosomes.

B. U6 can function as a branch acceptor and even as a 5' exon when mutations prevent it from correctly positioning the 5' splice site within the spliceosome.

Summary of Site-Directed Block Mutagenesis of Synthetic U6 snRNAs

Ascaris lumbricoides extracts form embryo (32-cell stage)

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Remove endogenous RNAs by micrococcal nuclease

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As in vitro functional reconstitution system of U6 snRNA

Total RNA was prepared from undeveloped Ascaris eggs

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5%-20% Sucrose gradients

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Pool snRNA fraction (4-8S)

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Titrate for ability to restore splicing activity to reconstitution system

Pack 5' biotinylated oligo complementary to U6 or

SL RNA in Streptavidin beads

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Mix with 4-8S RNA

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Primer extension analysis for U6, SL RNA or both was depleted

Ascaris extracts, ATP, PEG, poly(A) and labeling substrates

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Denaturing polyacrylamide gels analysis

Fig. 1 Build Functional Reconstitution of U6 snRNA in vitro assay system

1 2 3 4 5 1 2 3 4 5

extras w/o endogenous snRNA M + + + + M + + + +

4-8S fraction - + - - - + - -

4-8S RNA w/o U6 snRNA - - + - - - + -

Synthetic U6 RNA - - - + - - - +

Fig. 2 Site-directed mutant template U6 snRNA were tested for

their ability to reconstitute Cis- and Trans-splicing

Mutant 3 : 5' stem loop mutation

Mutant 6 : 8 bp upstream of conserved sequence, AAUU -> UUAA

Mutant 9: Change the first 4 bases of conserved ACAGAG box

in the central domain of U6

Mutant 10: Change 3' AG of universally conserved ACAGAG box

in the central domain of U6

Mutant 11,12: disrupt both the stem I U4-U6, interaction,

and the helix I U2-U6 interaction

Fig. 3 4% and 8% polyacrylamide gels to analysis the labeled pre-mRNA

in reconstitution Cis and Trans-splicing reactions

WT : had unexpected 2 intermediates between 2nd and 3rd step in Trans-splicing

Mutant 3: had unexpected 4 intermediates between 2nd and 3rd step in Trans-splicing

Mutant 6: Cis-splicing:

Efficiency of splicing was reduced.

* A new RNA species w/ retarded mobility appeared.

Trans-splicing:

Normal splicing was blocked.

Had a different slowly migrating RNA than

predicted Y-branched exon intermediate.

Mutant 9: Only had a relatively minor effect on splicing

8%* Both conserved sequence mutation appeared a new RNA species

Mutant 10: Block strongly the second step of Cis and Trans splicing

Mutant 11,12: Fail to support either Cis or Trans-splicing

Individual RNA was incubated with HeLa cell S100 cytoplasmic extract

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Debranching enzyme was specific to cleavage the single 2'5' phosphodiester bond in RNA lariats

Debranching of unusual migrating RNA in Fig. 3

Mutant 6 branched molecule was composed of 2 separated RNAs]

Debranching mutant 9,10 unusual migrating RNA yielded molecule that migrated faster than the substrate. These RNAs have internal branch points.

Fig. 4 4% polyacrylamide gels to analysis the labeled SL RNAs

in reconstitution Trans-splicing reactions

WT: Only one of two branched intermediates contains SL RNA

Mutant 3: Only one of four branched intermediates contains SL RNA

Mutant 6 : Intermediates and products were observed background levels

Mutant 9: Unusual mobility RNAs were not evident.

These molecules do not contain SL RNA sequence.

Mutant 10: A strong block to the second step of the reaction

Mutant 12: No Trans-splicing

These unusual migration intermediates did not involved branching to the SL RNA.

After debranching analysis, these branched RNA did not interact covalently w/ splicing substrate.

Individual RNA was incubated with specific snRNA or SL RNA

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Complementary double strand RNAs were digested by RNAase H

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Gel mobility shift assay

Complementary to snRNAs and RNAase H digestion

Mutant 6: The unusual migrating branched RNA only contained U6 snRNA.

Mutant 3: These 3 extra intermediates only contained U6 snRNA

WT: One extra intermediates only contained U6 snRNA

These unusual branched RNA molecules resulted from branch point attach upon U6 snRNA itself.

Fig. 5 4% polyacrylamide gels to analysis the incorporation of labeled U6 snRNAs

in reconstitution Cis and Trans-splicing reactions

(a, a') These unusual migrating RNA did incorporate to labeled U6 snRNA.

(c) Using RT-PCR to establish the more intense band that represent a spliced product, i.e. the liberated 5' exon of U6 is ligated to the exon in the Trans-splicing substrate at the proper position.

(b) The fainter band represents the Y-intron product of the 2nd step of Trans-splicing.

(b') Represents the Y intron product in 2nd step of Cis-splicing reaction, i.e. the 5' exon of U6 is joined to the downstream exon of the Cis-splicing substrate.

Fig 6 Debranching to map site of branch formation between U6 snRNAs and the

Trans-splicing pre-mRNA substrate

(A) After treatment w/ HeLa cell S100 extract, discrete fragments of U6 were release

from each branched intermediates.

(B) Use primer extension and two dimensional RNAase T1 fingerprint assays to map

the sites of branching more accurately and to establish which nucleotides initiated

branching.

Two adjacent adenosines 18nt and 19nt upstream of the Trans-splicing acceptor

site were used w/ equal frequency as branch points to U6, the branch point are

used in Trans-splicing to the SL RNA.

Fig. 7 Depletion of the SL RNA prior to reconstitution greatly reduced Trans-splicing

but did not effect Cis-splicing

Fig 7

Labeled Labeled Labeled

substrate U6 Mutant 6

|------| |-------| |----|

12 34 56 78 910

SL RNA in 4-8S RNA + - + - - - - - - -

U6 RNA in 4-8S RNA + + + + - - - - - -

Synthetic SL RNA - - - - +- +- + -

WT U6 RNA - - - - ++ - - - -

Mutant 6 U6 RNA - - - - - - ++ - -