Ch. 19: RNA Splicing and Processing

splicesome

*A complex that is required for RNA splicing, formed by snRNPs and additional protein factors. *The five snRNPs involved in splicing are U1, U2, U5, U4, and U6. *The ______ is ~12 MDa. Five snRNPs account for almost half of the mass. *The ______ is a large body, greater in mass than the ribosome.

Describe and diagram in detail the process of removing introns in the nuclei of eukaryotic cells that involves spliceosomes (lariat method).

*Pre-mRNA Splicing Proceeds Through a Lariat *Splicing requires the 5′ and 3′ splice sites and a branch site just upstream of the 3′ splice site. *The branch sequence is conserved in yeast but less well conserved in multicellular eukaryotes. *A lariat is formed when the intron is cleaved at the 5′ splice site, and the 5′ end is joined to a 2′ position [3' OH tied up in phosphodiester bonds, branch uses 2'] at an A at the branch site in the intron. *The intron is released as a lariat when it is cleaved at the 3′ splice site, and the left and right exons are then ligated together. *Splicing occurs in two stages. First the 5' exon is cleaved off, and then it is joined to the 3' exon.

If a primary RNA transcript contains 5 introns, what can be predicted about the order of their removal? What is the likely basis that determines this?

*Predict to be removed in order of transcription (5' - 3') because splicing can begin during transcription *Splicing Is Temporally and Functionally Coupled with Multiple Steps in Gene Expression *Splicing can occur during or after transcription. *The transcription and splicing machineries are physically and functionally integrated. *Splice Sites Are Read in Pairs *Splicing depends only on recognition of pairs of splice sites. *All 5′ splice sites are functionally equivalent, as are all 3′ splice sites. *Pre-mRNA splicing has long been recognized to take place cotranscriptionally, though the two reactions can take place separately in vitro and have been studied as separate processes in gene expression. Major experimental evidence supporting cotranscriptional splicing came from the observations that many splicing events are completed before the completion of transcription. In general, introns near the 5′ end of the gene are removed during transcription, but introns near the end of the gene can be processed either during or after transcription. *Introns are removed from the pre-mRNA transcript by splicing to give an mRNA that is colinear with the polypeptide product.

heterogeneous nuclear RNA (hnRNA)

*RNA that comprises transcripts of nuclear genes made by RNA polymerase II; it has a wide size distribution and low stability. *[ hnRNP - The ribonucleoprotein form of ______, in which the ______ is complexed with proteins. *Pre-mRNAs are not exported until processing is complete; thus they are found only in the nucleus}

Diagram and describe an example of where alternative RNA splicing is used in nature to regulate gene expression.

*Sex determination in Drosophila involves a series of alternative splicing events in genes encoding successive products of a pathway. *Sex determination in D. melanogaster involves a pathway in which different splicing events occur in females.

How are intron margins defined (consensus sequences)? What can be said about the order of intron removal from hnRNAs? What commonly happens if a right junction in deleted from an internal intron of an hnRNA? What does this demonstrate?

*The GU-AG rule (originally called the GT-AG rule in terms of DNA sequence) describes the requirement for these constant dinucleotides at the first two and last two positions of introns in pre-mRNAs *Minor introns exist relative to the major introns that follow the GU-AG rule; minor introns follow a general AU-AC rule with a different set of consensus sequences at the exon-intron boundaries. *Splicing Is Temporally and Functionally Coupled with Multiple Steps in Gene Expression *Splicing can occur during or after transcription. *The transcription and splicing machineries are physically and functionally integrated. *Splicing is connected to mRNA export and stability control. *Splicing in the nucleus can influence mRNA translation in the cytoplasm. *heterogeneous nuclear RNA (hnRNA) - RNA that comprises transcripts of nuclear genes made by RNA polymerase II; it has a wide size distribution and low stability *hnRNP - The ribonucleoprotein form of hnRNA in which the hnRNA is complexed with proteins. Pre-mRNAs are not exported until processing is complete; thus, they are found only in the nucleus.

pre-mRNA

*The nuclear transcript that is processed by modification and splicing to give an mRNA. *______s are not exported until processing is complete; thus they are found only in the nucleus

snRNP

*The splicing apparatus contains both proteins and RNAs (in addition to the pre-mRNA). The RNAs take the form of small molecules that exist as ribonucleoprotein particles. *Both the nucleus and cytoplasm of eukaryotic cells contain many discrete small RNA types. *Those restricted to the nucleus are called small nuclear RNAs (snRNAs; snurps) (several of them are involved in splicing or other RNA processing reactions); *those found in the cytoplasm are called small cytoplasmic RNAs (scRNAs) (and sometimes are also found in the nucleus). *In their natural state, they exist as ribonucleoprotein particles (______s and scRNPs). *Colloquially, they are sometimes known as snurps and scyrps, respectively.

Describe and diagram the process utilized to remove group I type self-splicing introns. How does this differ from group II self- splicing introns?

*Three classes of splicing reactions proceed by two transesterifications. First, a free -OH group attacks the exon 1- intron junction. Second, the -OH created at the end of exon 1 attacks the intron-exon 2 junction. *Group II brings its own -OH

Describe and diagram the process of removing an intron from a tRNA primary transcript. Provide the names of all enzymes and necessary cofactors. Where are these introns generally located?

*tRNA Splicing Involves Cutting and Rejoining in Separate Reactions *RNA polymerase III terminates transcription in a poly(U)4 sequence embedded in a GC-rich sequence. *tRNA splicing occurs by successive cleavage and ligation reactions. *The intron in yeast tRNAPhe base pairs with the anticodon to change the structure of the anticodon arm. *An endonuclease cleaves the tRNA precursors at both ends of the intron. *Release of the intron generates two half-tRNAs with unusual ends that contain 5′-OH hydroxyl and 2′,3′-cyclic phosphate. *The 3' and 5' cleavages in S. cerevisiae pre-tRNA are catalyzed by different subunits of the endonuclease. *The 5′-OH end is phosphorylated by a polynucleotide kinase, the cyclic phosphate group is opened by phosphodiesterase to generate a 2′-phosphate terminus and 3′-OH group, the exon ends are joined by an RNA ligase, and the 2′-phosphate is removed by a phosphatase. *Splicing of tRNA requires separate nuclease and ligase activities.

What is trans-splicing? Diagram and describe an example of where this occurs in nature.

*trans-Splicing Reactions Use Small RNAs *Splicing reactions usually occur only in cis between splice sites on the same molecule of RNA. *trans-splicing occurs in trypanosomes and worms where a short sequence (SL RNA) [5' Leader Sequence (SL)] is spliced to the 5′ ends of many precursor mRNAs. *SL RNAs have a structure resembling the Sm-binding site of U-snRNAs. *Figure 19.26: Splicing usually occurs only in cis between exons carried on the same physical RNA molecule