RNA splicing

  1. The coding sequence of a protein-coding gene is a series of three ______ ______ that specifies the linear sequence of _____ _____ in its polypeptide product.
    • nucleotide codons
    • amino acid
  2. What is the difference between the coding sequences in bacteria or phages and eukaryotes?
    • Bacteria have contiguous or continuous coding sequences, the codon for one amino acid is immediately adjacent to the codon for the next amino acid in the polypeptide chain. 
    • In eukaryotic genes, the coding sequence is periodically interrupted by stretches of non coding sequence
  3. Many _______ genes are thus mosaics, consisting of blocks of coding sequences separated from each other by blocks of non-coding sequences. The coding sequences are called ______ and the intervening sequences are called _____.
    • eukaryotic 
    • exons 
    • introns
  4. Once transcribed into an RNA transcript, the _______ must be removed and the _____ joined together to create the mRNA for that gene. In fact, technically, the term ______ applies to any region retained in a mature RNA, whether or not it is.
    • introns 
    • exons 
    • exon
  5. Name three non coding exon regions
    • 5' & 3' untranslated regions of mRNA
    • all portions of spliced, stable non-coding RNAs
    • regions that give rise to functional RNAs (miRNAs)
  6. The number of introns found within a gene varies enormously from ____ in the case of most intron containing yeast genes and few human genes, to _____ in the case of the chicken proα2 collagen gene, to as many as ____ in the case of titin gene of humans.
    • one 
    • 50
    • 363
  7. Clearly, the average number of introns per gene ______ from simple single-celled eukaryotes, such as ______, through higher organisms such as worms and flies all the way up to ______.
    • increases 
    • yeasts
    • humans
  8. Indeed, introns are very often much _______ than the exons they separate. Thus, for example, exons are typically on the order of ______ nucleotides, whereas introns (although they too can be ____)  can be as long as _______ nucleotides.
    • longer 
    • 150 nucleotides
    • short
    • 800,000 nucleotides
  9. Like the uninterrupted genes of prokaryotes, the split genes of eukaryotes are ________ into a single RNA copy of the entire gene, the primary transcript for a typical euarotic gene contains ______ as well as ______.
    • transcribed 
    • introns 
    • exons
  10. Because of the length of and number of introns, the ______ _____ (____) can be very long
    primary transcript (pre-mRNA)
  11. The primary transcripts of intron-containing genes must have their ______ removed before they can be translated into ______. The process of removal is called ______ _______.
    • introns
    • proteins
    • RNA splicing
  12. RNA splicing converts the ______ into _____ _____ and must occur with great precision to avoid the _____ or ______, of even a single nucleotide at the sites at which the exons are joined.
    • pre-mRNA
    • mature RNA
    • loss or addition
  13. Lack of precision in splicing, for example gaining or losing bases between two exons, would throw the reading frames of exons out of register. What does this entail?
    Downstream codons would be incorrectly selected and the wrong amino acids incorporated into proteins.
  14. Some pre-mRNAs can be spliced in more than one way. Thus, mRNAs containing different selections of exons can be generated from a given _____. This process is called ______ ______ and it enables a gene to give rise to more than one _______ product.
    • pre-mRNA
    • alternative splicing
    • polypeptide
  15. The different forms of polypeptide products arising from alternative splicing are called _______. It is estimated that ____ or more of the protein coding genes in the human genome are spliced in _______ ways to generate more than one _______.
    • isoforms 
    • 90%
    • alternative 
    • isoform
  16. Alternative splicing is often a regulated process, with different ______ being produced in response to different _______ or in different ____ types
    • isoforms
    • signals
    • cell
  17. The borders between introns and exons are marked by specific _______ ________ within the premRNAs which delineate where ______ will occur
    • nucleotide sequences
    • splicing
  18. The _____-_____ boundary at the 5' end of the intron is marked by a sequence called the __ ____ _____. The ______-_____ boundary at the 3' end of the intron is marked by the __ _____ _____. These sites are sometimes referred to as the ______ and the ______ sites respectively.
    • exon-intron 
    • 5' splice site 
    • intron-exon 
    • 3' splice site 
    • donor 
    • acceptor
  19. The third sequence necessary for splicing is called the ______ ______ or ______ ______. It is found entirely within the _____, usually close to its ___ end, and is followed by a _______ tract
    • branchpoint site or branchpoint sequence 
    • intron
    • 3'
    • polypyrimidine tract (Py tract)
  20. Highly conserved nucleotides are all found within the ______ itself. Perhaps not surprisingly, because the sequence of most exons, in contrast to the _____, is constrained by the need to encode the specific ______ ______ of the protein product.
    • intron
    • intron
    • amino acids
  21. An intron is removed through two successive __________ reactions in which ________ linkages within the pre-mRNA are broken and new ones are formed.
    • transesterification 
    • phosphodiester
  22. The first reaction is triggered by the ______ of the conserved A at the ______ site. This group acts as a _______ to attack the ________ group of the conserved G in the _____ _____ site.
    • 2'OH 
    • branch site
    • nucleophile 
    • phosphoryl 
    • 5' splice site
  23. Note that the 5' exon is a ______ group in the first transesterification reaction. In the second reaction, the 5' exon (more precisely, the newly liberated _____ of the 5' exon) reverses its role and becomes a ______ that attacks the _______ group at the 3' splice site.
    • leaving 
    • 3'OH
    • nucleophile 
    • phosphoryl
  24. The two consequences of the second transesterification reaction
    • First and most importantly, it joins the 5' and 3' exons; thus, this is the step in which the two coding sequences are actually spliced together
    • Second, this same reaction liberates the intro, which serves as a leaving group
  25. Because the 5' end of the intron had been joined to _________ in the first transesterification reaction, the newly liberated intron has the shape of a _____.
    • branchpoint A
    • lariat
  26. In the two reactions steps, there is no net gain in the number of chemical bonds, explain.
    two phosphodiester bonds are broken and two new ones are made
  27. Since the two reaction steps are just a question of shuffling bonds, no ______ input is demanded by the chemistry of this process. However, a large amount of ____ is consumed during the splicing reaction. Why is this necessary?
    • energy
    • ATP
    • Necessary for properly assembling and operating the splicing machinery
  28. RNA splicing is performed by a large complex called the _______
    spliceosome
  29. The transesterification reactions are mediated by a huge molecular machine called the ________. This complex comprises about _____ proteins and _____ RNAs. It is similar in size to a ______, the machine that translates mRNA into protein.
    • spliceosome
    • 150
    • 5
    • ribosome
  30. In performing a even a single splicing reaction, the spliceosome ________ several molecules of ATP. Strikingly, it is believed that many of the functions of the spliceosome are performed by its _____ components rather than the ______. This is reminiscent of the _______
    • hydrolyzes
    • RNA 
    • protein
    • ribosome
  31. What is the likely role of RNA components in the spliceosome
    RNAs locate the sequence elements at the intron-exon borders and likely participate in catalysis of the splicing reactions itself
  32. Name 5 RNAs of the spliceosome. They are collectively called _____ ____ ______. Each of these RNAs is between 100 & 300 nucleotides long in most _______ and is complexed with several ______
    • U1,U2,U4,U5 & U6
    • small nuclear RNAs (snRNAs)
    • eukaryotes
    • proteins
  33. The protein complexes of the spliceosome are called _____ _____ ________ _______
    small nuclear ribonuclear proteins (snRNPs "snurps")
  34. The spliceosome is the large complex made up of _______, but the exact makeup differs at different stages of the splicing reaction. Different ______ come and go at different times, each performing ______ functions in the reaction. There are also many ______ within the spliceosome that are not part of the ______.
    • snRNPs
    • snRNPs
    • particular 
    • proteins
    • snRNPs
  35. The three roles of snRNPs in splicing
    • They recognize the 5' splice site and the branch site
    • They bring those sites together as required
    • They catalyze or help to catalyze the RNA cleavage and joining reactions
  36. For snRNPs to perform their roles, which interactions are important
    • RNA-RNA
    • RNA-protien
    • Protein-Protein
  37. Where do RNA-RNA interactions occur?
    • Within individual snRNPs
    • Between snRNPs
    • Between snRNPs and the pre-mRNA
  38. Some non-snRNps are involved in ______, for example, the U2AF (U2 auxillary factor), recognizes the _________ ______/ ______ _____ site and in the initial step of the splcing reaction helps another _________-______ protein bind to the branch site.
    • splicing
    • polypyrimidine (Py) tract/ 3' splice site 
    • branchpoit-binding protein (BBP)
  39. Other proteins involved in the splicinng reaction include ____-_______ ______ which help load snRNPs onto the mRNA, and ______-___ _______ proteins, which use their ATPase activity to dissociate given RNA-RNA interactions. What is the result of this?
    • RNA-annealing factors
    • dead-box helicase proteins 
    • Allows alternative pairs to form and thereby drives the rearrangements that occur through the splicing reaction.
  40. What is required to remove spliced mRNA from the spliceosome and trigger spliceosome disassembly
    dead-box helicase proteins
  41. Story: Tell the cannonical splicing pathway of assembly (pt 1 E through A complex) (4)
    • Initially, the 5' splice site is recognized by the U1 snRNP (using base pairing between its snRNA and the pre-mRNA)
    • U2AF is made up of two subunits, the larger of which (65) binds to the Py tract and the smaller (35) binds to the 3' splice site
    • The former subunit interacts with BBP (SF1) and helps that protein bind to the branch site (this arrangement of proteins and RNA is called the early (E) complex
    • U2 snRNP then binds to branch site, aided by U2AF and displacing BBP (SF1). This is called the A complex
  42. The base pairing in the A complex between the ______ and the _____ _____ is such that the ____ ____ A residue is extruded from the resulting stretch of double-helical RNA as a single bulge. This A residue is thus unpaired and available to react with the _______ _____
    • U2 snRNA & branch site
    • branch site
    • 5'splice site
  43. Story: Tell the cannonical splicing pathway of assembly pt 2 (5)
    • The next step is a rearrangement of the A complex to bring together three splice sites. 
    • This is achieved as follows: the U4 and U6 snRNPs, along with the U5 snRNP, join the complex.
    • Together, these three snRNPs are called the tri-snRNP particle, within which the U4 and U6 snRNPs are held together by complementary base pairing between their RNA components, and the U5 snRNP is more loosely associated through protein-protein interactions. With the entry of the tri-snRNP, the A complex is converted into the B complex
    • Next, U1 leaves the complex, and U6 replaces it at the 5' splice site. This requires that base pairing between the U1 snRNA and the pre-mRNA be broken, allowing the U66 RNA to anneal with the same region 
    • This completes the assembly pathway
  44. Story: Tell the cannonical splicing pathway of catalysis
    • U4 is released from the complex allowing U6 to interact with U2 (This is accomplished through RNA:RNA base pairing)
    • This arrangement, called the C complex, produces the active site
    • The same rearrangement also ensures that the substrate RNA is properly positioned to be acted upon.
  45. Expand on what happens when the C complex is formed and an active site is produced
    The rearrangement brings together within the spliceosome those components (believed to be solely regions of the U2 and U6 RNAs) that together form the active site.
  46. It is striking not only that the active site is primarily formed of _____, but also that it is only formed at this stage of spliceosome assembly. Presumably, this strategy _______ the chance of abberant splicing.
    • RNA
    • lessens
  47. Linking the formation of the active site to the successful completion of earlier steps in the spliceosome assembly makes it highly likely that the active site is only available at which sites?
    legitimate splice sites
  48. Formation of the active site juxtaposes the _____ _____ of pre-mRNA and the _____ ______, facilitating the first transesterification reaction. The second reaction is between the ____ & ____ splice sites and is aided by the ___ snRNP, which helps to bring the two _____ together. The final step involves releases of the ____ product and the _______.
    • 5'splice site 
    • branch site
    • 5' & 3' splice sites
    • U5 
    • exons 
    • mRNA 
    • snRNPs
  49. The average human gene has seven or eight exons and can be spliced in three alternative forms. But there is one human gene with ____ exons and one drosophila gene that can be spliced in ______ alternative ways
    • 363
    • 38,000
  50. If the snRNPs had to find the correct 5' and 3' splice sites on a complete RNA molecule and bring them together in the correct pairs, unaided,  it seems inevitable that many ______ would occur
    errors
  51. Splice-site recognition is prone to two kinds of errors, name them
    • First, splice sites can be skipped, with components bound at, for example, a given 5'splice site pairing with those at a 3' site beyond the correct one
    • Second, other sites, close in sequence but not legitimate splice sites could be mistakenly recognized (recall that the splice site consensus sequences are rather loose)
  52. State an example of the second splice-site recognition error
    components at a given 5' splice site might pair with components bound incorrectly at such a pseudo 3' splice site
  53. Two ways in which accuracy of splice-site selection can be enhanced
    • First, while transcribing a gene to produce the RNA, RNA polymerase II carries with it various proteins with roles in RNA processing (including proteins involved in splicing)
    • A second mechanism guards against the use of incorrect sites by ensuring that splice sites close to exons (more likely to be authentic) are recognized preferentially
  54. In the first mechanism, when a 5'splice site is encountered in the newly synthesized RNA, the factors that recognize THAT site are transferred from _______ _______-_______ "tail" onto the _____. Once in place, the 5'splice site components are poised to interact with those other factors that bind to the next 3' splice site to be synthesized. What is the result?
    • polymerase carboxy-terminal tail
    • RNA
    • The correct 3'splice site can be recognized before any competing sites further downstream have been transcribed, this contrascriptional loading process greatly diminishes the likelihood of exon skipping
  55. In the second mechanism, so called SR (_____-_____ rich) proteins bind to sequences called exonic splicing enhancers (ESEs) within the _____. SR proteins bound to these sites recruit the _____ _____ to the nearby splice sites. In this way, the machinery binds more ______ to those nearby splice sites than to incorrect sites not _____ to the exon.
    • serine-arginine rich
    • exon
    • splicing machinery
    • efficiently 
    • close
  56. By recruiting splicing factors to each side of a given exon, this process encourages the so called exon definition. Meaning spliceosome components are recruited around ______ initially, rather than around the _____ to be removed. Subsequently, components near one _____ will pair with those near an adjacent _____ to eliminate the intervening ______.
    • exons
    • introns
    • exon
    • exon
    • intron
  57. SR proteins are essential for ______ they not only ensure the accuracy and efficiency of constitutive splicing, but also regulate ______ ______. They come in many varieties, some controlled by _______ signals others ______ active. Some are expressed preferentially in certain ____ types and control ______ according to said types.
    • splicing
    • alternative splicing 
    • physiological signals
    • constitutively active
    • cell types
    • splicing
  58. In alternative splicing, exons can be deliberately ______, and a given exon is joined to one further downstream. In some cases, two exons carried on different RNA molecules can be spliced together in a process called ____-_____.
    • skipped 
    • trans-splicing
  59. Although generally rare, trans-splicing occurs in almost all of the _____ of trypanosomes (for example the ______). Trans-splicing uses the same spliceosomal machinery as normal cis-splicing, except for _____ which at least in worms, is not needed for trans-splicing
    • mRNAs
    • nematode
    • U1
  60. Higher eukaryotes (including mammals, plants etc) use the major splicing machinery to direct splicing of the majority of their ___-____. But in these organisms, unlike in ______, some are spliced by an ______ ow abundance form of the spliceosome.
    • pre-mRNA
    • yeastss
    • alternative
  61. The rare form of alternative splicing, in eukaryotes, contains some components common to the major spliceosome, but it contains other unique components as well. ____ & ____ components of the alternative spliceosome have the same roles in the splicing reaction as U1 and U2 of the major form, but they recognize distinct _______. ____ & ____ have equivalent counterparts in both spliceosome forms although these snRNPs are _______ they share the same names. Finally, the identical ____ component is found in both major and the alternative (minor) spliceosome.
    • U11 & U12
    • sequences
    • U4 & U6
    • distinct
    • U5
  62. The minor spliceosome recognizes rarely occurring ______ having consensus sequences distinct from sequences of most ___-____ ______.
    • introns
    • pre-mRNA introns
  63. It should be emphasized that although these introns are rare, they are ______ distributed (approx. 800 human genes contain at least one _____ _____). Furthermore, mutations in minor snRNAs have recently been found to underlie some rare _____ ______ diseases
    • widely
    • minor intron
    • human genetic
  64. Why is the minor form of the spliceosome also known as the AT-AC spliceosome?
    Because the termini of the originally identified rare introns contain AU at the 5' spliice site and AC at the 3' splice site (in RNA or AT and AC in DNA)
  65. Later it transpired that many introns spliced by the AT-AC spliceosome have a GT-AG termini (like mainstream introns), but otherwise their consensus sequences are _______ from those of the ______ pathway
    • distinct 
    • major
  66. Alternative splicing is sometimes used as a way of generating _______, with alternative forms being generated stochastically. However, in many cases, the process is regulated to ensure that different ______ ______ are made in different cell types or in response to different conditions
    • diversity
    • protein products
  67. Alternative splicing can occur in a number of ways, exons can be ______, _______ or introns can be ______ in the mature message.
    • extended 
    • skipped (deliberately)
    • retained
  68. How can exons be extended
    by selecting an alternative downstream 5', our upstream 3', splice site
Author
chikeokjr
ID
331094
Card Set
RNA splicing
Description
Ch 14
Updated